Invitrogen Flow cytometry capabilities guide

Flow cytometry
capabilities guide

Conjugated Cell health Flow RNA Everyday Flow cytometry
antibodies essentials instrument

Don’t settle for average—
real insight starts with single cells

Push the boundaries of cell analysis

Flow cytometry is an important technique central to many of the fastest-growing
areas of life science research, such as immuno-oncology, antibody therapeutic
development, and gene editing, because it empowers researchers to study biology
at the single-cell level.

At Thermo Fisher Scientific, we are committed to accelerating your science by
providing a comprehensive suite of solutions for the analysis of cells and their
functions. Behind this commitment is an incredible team of scientists developing
and supporting our innovative instrumentation and products, such as the Invitrogen™
Attune™ NxT Flow Cytometer, Invitrogen™ eBioscience™ flow cytometry antibodies,
and Invitrogen™ flow cytometry assays and reagents.

We are focused on advancing meaningful discoveries and partnerships to make
tools for cellular analysis widely accessible, affordable, and powerful for all life
science scientists. When you’re on your quest for significant breakthroughs, we
know that you never settle for average, and neither will we.

Contents

Flow cytometry workflow capabilities 4

Antibodies, assays, and reagents

Antibodies 6

Assays 9

Beads for instrument controls and standards 14

Sample preparation buffers and reagents 16

Instrumentation 18

Flow cytometry Flow cytometry Flow cytometry
antibodies, assays, and reagents instrumentation education and support

Flow cytometry workflow capabilities

Uniquely positioned to address your workflow needs

Flow cytometry enables the simultaneous, multiparametric However, ensuring that experimental conditions are
analysis of proteins, gene expression, and cell functions consistent and data are reproducible can be challenging
such as viability, cell cycle, apoptosis, and proliferation. in flow cytometry. In response to this dilemma, we have
This technology makes it possible to obtain a statistically developed high-quality instruments and products that
relevant amount of data from a heterogeneous cell are validated* and optimized by our flow cytometry R&D
population. Whether you are identifying cell subpopulations, team to help facilitate reproducible results and to reduce
analyzing cells at the single-cell level, or investigating what error, thereby saving your laboratory time and money.
is going on within them, flow cytometry offers significant Additionally, using products and systems that have been
advantages over other applications. designed to work together takes some of the guesswork
out of flow cytometry and helps enable better, more
reproducible data.

* The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the
antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.

4

Antibodies, assays, and reagents— Beads for instrument controls and standards—
Our diverse collection of antibodies, Flow cytometers are designed to perform quantitative
assays, beads, and buffers support leading measurements on individual cells and other particles with
research areas. high precision, speed, and accuracy, making instrument
calibration a critical step in flow cytometer performance
Antibodies— and data integrity. Experimental controls for the analysis of
Over 10,000 conjugated antibodies are specifically fluorescently labeled cells include compensation, absolute
developed, validated*, and manufactured for flow cell counts, and live vs. dead cells determination.
cytometry applications. Multiple fluorophore format
options are available to help you build and expand your Sample preparation buffers and reagents—
panels as your research demands. An essential part of Quality data requires quality starting material. Achieve
evaluating any biological system is the ability to mimic the best possible results with highly referenced sample
or inhibit relevant pathways. This can be done with preparation reagents for flow cytometry, including
the use of Invitrogen™ eBioscience™ Functional Grade intracellular and transcription factor staining buffer sets,
antibodies for activation, blocking, or neutralization cell lysis and preservation assays, blocking reagents, and
studies, as well as recombinant proteins. These are all in magnetic cell isolation beads.
appropriate preservative-free buffers with extremely low
endotoxin levels to prevent any nonspecific side effects. Flow cytometry instrumentation—
thermofisher.com/flowantibodies Instruments like the Attune NxT Flow Cytometer
have helped expand the boundaries of flow
Assays— cytometric analysis. The Attune NxT system was
We offer flow cytometry assays and reagents that span a developed with the goal of removing barriers to classical
broad range of research applications and areas, including flow cytometry to better enable new progress in this field.
cell health and function assays as well as RNA detection This includes increased data fidelity with faster acquisition
assays. thermofisher.com/flow-assays rates and improved fluidics with acoustic focusing and
resistance to clogging, thereby enlarging the scope and
RNA detection by flow cytometry— power of flow cytometry to areas such as cancer and
The Invitrogen™ PrimeFlow™ RNA Assay reveals the synthetic biology. thermofisher.com/attune
dynamics of RNA and protein expression within individual
cells, facilitating unprecedented analysis of their correlation Flow cytometry education and support—
as cells change over time or in response to stimuli. This Our Thermo Scientific™ and Invitrogen™ portfolios
novel assay employs a proprietary fluorescence in situ of instruments are supported by an extensive
hybridization (FISH) and branched DNA (bDNA) signal global team of experienced scientists and
amplification technique, enabling simultaneous detection engineers, who all adhere to a tenet of unrelenting reliability
of as many as four RNA transcripts in a single cell using a for service and support. We are also committed to
standard flow cytometer. thermofisher.com/primeflow education. Expand your understanding with our curated
collection of Flow Cytometry Resource Library of technical
application notes, publications, videos, webinars, and
scientific posters for flow cytometry. Find out more at
thermofisher.com/flowlearning

* The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody
can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.

5

Antibodies, assays, and reagents

Antibodies

With the addition of the eBioscience antibodies, we offer From subset identification of heterologous cell populations
a portfolio of over 10,000 primary antibodies specifically (Figure 1) to rare-event detection, our fluorescently
developed, validated, and manufactured for flow cytometry conjugated primary antibodies can help answer complex
applications. This catalog includes traditional and unique questions, in less time and with less sample than using
antibody specificities for all of your flow cytometry research other approaches. Regardless of your instrument, we
needs, from the group that delivered the first commercially have labels designed to help you get the most out of every
available antibodies for key immunology targets, including sample and every flow cytometry run.
Foxp3, RORγt, TIGIT, CD223 (LAG3), and EOMES.
Search for your primary antibody conjugate at
Our flow cytometry antibodies are conjugated to numerous thermofisher.com/flowantibodies
different fluorophores to allow for flexibility, ease, and
expansion of your panel needs. In addition to the traditional Flow conjugated antibodies for:
fluorophores such as PE, FITC, and APC, we offer three
additional families of fluorophores to greater expand the • CD markers • Immuno-oncology
depth of the flow cytometry antibody portfolio. These
include Invitrogen™ Alexa Fluor™, eFluor™, and Super Bright • Transcription factors • Oncology
dyes. These families of fluorophores, combined with our
broad portfolio of biological content, more easily enable • Cytokines, chemokines, and • Apoptosis
dye selection for optimized multicolor antibody panel growth factors • Cell cycle
design for flow cytometry. • Stem cells
• Phosphospecific cell signaling

• Immunology

CD20 PE-Cyanine5.5 CD4+ CD25 Super Bright 600 CD25+ Foxp3 FITC Treg cells
CD4 eFluor 506
CD3+ CD8+ CD25– Normalized to Mode
CD4 eFluor 506
CD3 Super Bright 645 CD8 APC-eFluor 780 CD4 eFluor 506

Th1 inflammatoryCD183 PE-eFluor 610 Mature/activated Treg cells
CD4 T cells T cells
CD4 T
TIGIT APC Foxp3 FITC helper cells TIGIT APC

CD4 eFluor 506 Subset name Count
CD25– 12245
CD25+ 883

!"LN-homing CD27 Super Bright 436
T cells

CD27 Super Bright 436

Panel: Anti-CD25 (clone BC96) Super Bright 600 Anti-CD185 (CXCR5) (clone MU5UBEE)
PE-Cyanine7
Anti-CD3 (clone OKT3) Super Bright 645 Anti-CD27 (clone O323) Super Bright 436
Anti-CD4 (clone RPA-T4) eFluor 506 Anti-Foxp3 (clone PCH101) FITC
Anti-CD8a (clone RPA-T8) APC-eFluor 780 Anti-CD183 (CXCR3) (clone CEW33D)
Anti-CD20 (clone 2H7) PE-Cyanine5.5 PE-eFluor 610 Anti-TIGIT (clone MBSA43) APC

Figure 1. Ten-color T cell subset panel. Human peripheral blood cells were aliquoted in the presence of Super Bright Staining Buffer, then surface-
stained with the indicated reagents. Samples were then fixed and permeabilized according to the Invitrogen™ eBioscience™ Foxp3/Transcription Factor
Staining Buffer Set protocol and stained with the indicated intracellular reagents. Analysis was performed to discriminate various T cell subpopulations.

6

Super Bright antibody conjugates

Brighter dyes for the violet laser Advantages of Super Bright conjugates
Featuring: • Provide improved detection of low-abundance antigens
• Super Bright 436 dye • Super Bright 702 dye
• Allow for more data acquisition using the violet laser,
• Super Bright 600 dye • Super Bright 780 dye freeing up other channels

• Super Bright 645 dye • Super Bright Staining Buffer • In some cases, require less compensation and have less
dye–dye interaction than comparable products
The Super Bright dyes are a line of bright fluorophores that
are optimized for use in flow cytometry and may allow for • Optimal choice for multicolor flow cytometry, as
better discrimination of dim cell populations. They allow they can have lower background than reagents from
greater utilization of the violet laser, enabling detection in other suppliers
the range of 436–780 nm. This provides greater signal
separation, and in some cases, reduced compensation. • Minimal fluorescence loss when exposed to
Named according to their emission wavelengths (Figure 2), formaldehyde fixative solution or ambient light
Super Bright antibody conjugates are also compatible
with standard fluorophores, compensation beads, viability Stability studies
stains, and other flow cytometry reagents. Stability studies indicate that all Super Bright conjugates
exhibit minimal loss of fluorescence when cells are
Super Bright 436 Super BrSiguhpte6r0B0righSt u6p4e5r BrighSt u7p0e2r Bright 780 exposed to formaldehyde fixative for up tp 3 days, or when
exposed to overnight to ambient light.
100
Comparative data
80 Super Bright antibody conjugates have been tested and
compared to other fluorophores for the violet laser, and in
60 many instances, provide superior brightness (Figure 3).

40

20

0

300 nm 400 500 600 700 800 900

Figure 2. Emission spectra of Super Bright polymer dyes. The black
bar indicates the excitation wavelength of the violet laser (405 nm).

A B CD E

CD19 (clone HIB19) CD8a (clone 53-6.7) CD8a (clone 53-6.7) CD4 (clone GK1.5) CD4 (clone RM4-5)

Super Bright 436 Super Bright 600 Super Bright 645 Super Bright 702 Super Bright 780
eFluor 450 Briliant Violet 605 Brilliant Violet 650 Brilliant Violet 711 Brilliant Violet 786
Brilliant Violet 421

Figure 3. Fluorescence intensity comparison. (A) Human peripheral blood cells were stained with a CD19 antibody conjugated to Super Bright 436,
Invitrogen™ eBioscience™ eFluor™ 450, or Brilliant Violet™ 421 dye, using the recommended volume per test. Mouse splenocytes were stained with either
CD8a antibody conjugated to (B) Super Bright 600 or Brilliant Violet 605 dye or (C) Super Bright 645 or Brilliant Violet 650 dye or (D) CD4 antibody
conjugated to Super Bright 702 or Brilliant Violet 711 dye or (E) Super Bright 780 or Brilliant Violet dye. For (B–D), all experiments were conducted at the
same antibody concentration.

7

Stimulation

An essential part of evaluating any biological system is the Ready-to-use stimulants
ability to mimic or inhibit relevant pathways, both at the
cellular and tissue level and in whole-organism models Rat IgG2a PE-eFluor 610
using antibodies, proteins, or small molecules. We offer an Rat IgG2a PE-eFluor 610
expansive list of high-quality cell stimulation products that Rat IgG2a PE-eFluor 610
include Functional Grade antibodies, recombinant proteins, IL-17A PE-eFluor 610
and ready-to-use stimulants (e.g., Invitrogen™ eBioscience™ IL-17A PE-eFluor 610
Cell Stimulation Cocktails (PMA and Ionomycin), Con A, IL-17A PE-eFluor 610
PHA-L) as well as Invitrogen™ Dynabeads™ magnetic beads
for T cell activation and expansion (Figure 4). Functional CD5 PerCP-eFluor 710
Grade antibodies are used for activation (e.g., anti-CD3 CD4 PerCP-eFluor 710CD5PerCP-eFluor710 CD4 PerCP-eFluor 710
and anti-CD28), blocking (e.g., anti–PD-1 and anti-TIGIT), or CD4 PerCP-eFluor 710CD4PerCP-eFluor710
neutralization (e.g., anti–IL-2 and anti–IFN-gamma) studies.
These reagents are provided in appropriate preservative- Figure 4. Splenocytes were cultured under Th17-polarizing conditions
free buffers with extremely low endotoxin levels. for 6 days then restimulated for 5 hours with the Cell Stimulation
Cocktail (plus protein transport inhibitors). Cells were intracellularly
stained with Anti-Mouse CD4 PerCP-eFluor 710 Antibody and Rat
IgG2a K Isotype Control PE-eFluor 610 Antibody (left) or Anti–Mouse/
Rat IL-17A PE-eFluor 610 Antibody (right) using the Intracellular Fixation
& Permeabilization Buffer Set and protocol. Viable cells, as determined
by an Invitrogen™ fixable viability dye in the lymphocyte gate, were used
for analysis.

T cell activation Nature Laboratory

Dendritic cell 2nd signal 2nd signal
CD80/
CD86 CD28 CD28 Anti-CD28

T cell CD3/TCR

MHC CD3/
TCR

1st signal Anti-CD3
1st signal

Activated
T cell

8

Assays

Apoptosis Cell proliferation
Understanding the mechanisms of cell death Cell proliferation and the characterization
and survival can represent a critical aspect of agents that either promote or retard cell
of toxicological profiling and drug discovery. proliferation are extremely important areas of cell
Invitrogen™ reagents and assays are designed to effectively biology and drug discovery research. We offer the latest
study changes in the plasma membrane, mitochondria, technology for measuring new DNA synthesis (Invitrogen™
caspase activity, and DNA fragmentation and chromatin Click-iT™ Plus EdU labeling kits), as well as reagents for
condensation as a result of apoptosis. For information assessing cell proliferation by dye dilution (Invitrogen™
about our apoptosis assays for flow cytometry, go to CellTrace™ cell proliferation kits, Table 1).
thermofisher.com/flow-apoptosis
For more information on cell proliferation, go to
Cell viability thermofisher.com/flow-cellproliferation
Cell viability assays can be used to simply
distinguish between live and dead cell A Lymphocyte gate B Viability gate
populations, to correlate with other cell functions
or treatments, or to exclude dead cell populations from FSC-A FSC-A
analyses to avoid false-positive results (Figure 5). We offer a
selection of Invitrogen™ viability dyes (Table 1) and assays to
choose from:

• Fixable viability dyes

• Nonfixable viability dyes SSC LIVE/DEAD Fixable Violet fluorescence
• Bacterial viability and vitality assays
• Yeast viability and vitality assays C D

Learn more about our viability assays at CD4-Cyanine5.5-PE fluorescence CD4-Cyanine5.5-PE
thermofisher.com/flow-cellviability fluorescence

Cell cycle CD8-Q705 fluorescence CD8-Q705 fluorescence
Flow cytometry, in conjunction with modeling
algorithms, provides a powerful tool to assess Figure 5. Exclusion of dead cells eliminates staining artifacts from
cells in the G0/G1 phase of the cell cycle versus analysis. After the application of a lymphocyte gate (A), live and dead
the S phase and G2/M phase, or to analyze polyploidy. cells were discriminated (B) using the LIVE/DEAD Fixable Violet Dead Cell
Invitrogen™ fluorescent dyes allow accurate cell cycle Stain Kit. Note the significant number of dead cells despite a scatter gate.
analysis in either live or fixed cell populations (Table 1). Subsequent analysis of dead cells (C) and live cells (D) shows the dramatic
difference in apparent phenotypes between the two cell populations.
For more information about our cell cycle products, go to Reprinted from Perfetto SP, Chattopadhyay PK, Lamoreaux L, et al. (2006)
thermofisher.com/flow-cellcycle J Immunol Methods 313:199–208, with permission from Elsevier.

9

RNA detection by flow cytometry

Expanding the capability of flow cytometry to Novel product applications:
measure RNA • Unmask gene expression heterogeneity at the
Flow cytometry, with its ability to acquire and analyze
millions of individual cells simultaneously, use multiplexing single-cell level
capabilities, and to detect both cell-surface and
intracellular proteins in a straightforward workflow, has • Correlate RNA and protein levels in the same cell
long been the standard for characterizing heterogeneous
cell populations. Nevertheless, flow cytometry has been • Detect noncoding RNA, including microRNA, in
constrained by the availability and adequacy of antibodies cellular subsets
to measure all analytes of interest. There are many
areas of research such as microRNA, viral transcripts in • Evaluate viral RNA in infected cells
infected cells, and antibody development in unique model
organisms that have not been able to utilize the power of • Analyze mRNA expression when antibody selection
flow cytometry and have historically required numerous is limited
disconnected experiments by the researchers to analyze
these cellular systems. New features:
• Analyze up to four RNA transcripts simultaneously
With the novel PrimeFlow RNA Assay, scientists can
now reveal the dynamics of RNA and protein expression • Detect microRNA (miRNA) by flow cytometry
simultaneously within millions of single cells (Figure 6).
This assay employs a proprietary FISH and branched DNA Assay technology
(bDNA) amplification (Figure 7) technique for simultaneous FISH is a powerful technique that allows specific
detection of up to four RNA transcripts labeled with Alexa localization of RNA targets in fixed cells. The basic premise
Fluor 488, Alexa Fluor 568, Alexa Fluor 647, and Alexa Fluor of the application relies on detecting nucleic acids through
750 dyes, in a single cell using a standard flow cytometer. sequential hybridization of nucleic acid probes that provide
RNA detection may be combined with intracellular and cell- gene expression information at the single-cell level.
surface antibody staining to elevate the understanding of Traditional FISH techniques are generally limited by high
single-cell dynamics to a new dimension. background and low sensitivity due to nonspecific binding
and inefficient signal amplification.

miR-146a Alexa Fluor 647
Arg1 mRNA Alexa Fluor 488
Cxcl13 mRNA Alexa Fluor 750
Retnla mRNA Alexa Fluor 568

F4/80 eFluor 450 F4/80 eFluor 450 F4/80 eFluor 450 F4/80 eFluor 450

Figure 6. PrimeFlow RNA Assay detection of miR-146a, Arg1 mRNA, Cxcl13 mRNA, and Retnla mRNA in mouse peritoneal cells.
C57BI/6 mouse resident peritoneal exudate cells were analyzed using the PrimeFlow RNA Assay. Cells were stained with Invitrogen™ eBioscience™
Anti-Mouse F4/80 eFluor 450 and Anti-Mouse CD11b PE-Cyanine7 antibodies, then fixed and permeablized using PrimeFlow RNA Assay buffers and
protocols. Cells were then hybridized to label RNA with Invitrogen™ Type 1 Human/Mouse miR146a Alexa Fluor 647, Type 4 Mouse Arg1 Alexa Fluor 488,
Type 6 Mouse Cxcl13 Alexa Fluor 750, and Type 10 Mouse Retn1a Alexa Fluor 568 target probes. Viable CD11b+ cells were used for analysis. Data show
that both small peritoneal macrophages (SPM, F4/80–) and large peritoneal macrophages (LPM, F4/80+) were positive for miR-146a. SPM expressed high
levels of Retn1a (Relm-alpha) mRNA, whereas LPM were positive for Cxcl13 mRNA and expressed low levels of Arg1 mRNA.

10

The PrimeFlow RNA Assay incorporates a proprietary In the PrimeFlow RNA Assay, target-specific probe sets
oligonucleotide probe set design and bDNA signal contain 20–40 oligonucleotide pairs that hybridize to the
amplification technology to analyze RNA transcripts by flow target RNA transcript. Signal amplification is achieved
cytometry. bDNA technology provides a unique approach through specific hybridization of adjacent oligonucleotide
to RNA detection and signal amplification by amplifying the pairs to bDNA structures, formed by pre-amplifiers,
reporter signal rather than the target sequence (e.g., PCR) amplifiers, and fluorochrome-conjugated label probes,
to help enable more consistent results than those obtained resulting in excellent specificity, low background, and high
using PCR-based assays. signal-to-noise ratios (Figure 8).

Principle of the PrimeFlow RNA Assay LLaabbeel Pl rporboebe
Development of the PrimeFlow RNA Assay is based upon AAmmpplifilifierer
proven and well-published ViewRNA™ technology designed PPrree-A-ammplpifileifirer
for microscopic analysis of RNA in cells and tissues. The RRNNAA
assay combines paired oligonucleotide probe design with
bDNA signal amplification to robustly detect up to four LLefetfOt loigliogo RighRtigohlitgOoligo
RNA transcripts at the single-cell level using a standard
flow cytometer. Figure 7. PrimeFlow branched DNA illustration.

Sample preparation Target hybridization Signal amplification Detection

Gene-specific Pre-amplifier Fluorescence-labeled probes
label extenders (LE) Amplifier Add labeled probes to cells

Label proteins with antibody Incubate cells with gene-specific Hybridize with
(optional) probe sets pre-amplifier and
amplifier DNA
Fix and permeabilize cells in
suspension Process cells using
a flow cytometer
Label intracellular proteins with
antibodies (optional)

Gene 1 CD8 mRNA Alexa Fluor 647
Gene 1

Gene G1ene G2Geennee21 Gene 1
Gene 1

Gene 2 Gene 2 Gene 2
Gene 1 Gene 2

GeGnen2e 1

Suspension Gceenlels2with fixed RNA CD8 PE-Cyanine7

Figure 8. The PrimeFlow RNA Assay workflow. The assay workflow contains several steps: antibody staining; fixation and permeabilization, including
intracellular staining, if desired; followed by target hybridization with a target-specific probe set containing 20–40 oligonucleotide pairs.

11

Table 1. Fluorophore and reagent selection guide for flow cytometry.

Excitation Common Attune NxT Recommended Viability dyes Viability dyes DNA content/ DNA content/ Apoptosis Cell Reactive Phagocytosis Fluorescent Other dyes
laser emission channel dyes (compatible (unfixed cell cycle cell cycle dyes proliferation oxygen dyes proteins BD Horizon
filters (nm) (nm)* with fixation) cells) dyes dyes species BUV395
(live cells) Annexin V, dyes (ROS) dyes
(fixed cells) Alexa Fluor BD Horizon
CellTrace Blue BUV496
379/28 NA 350 Click-iT Plus EdU
Annexin V, BD Horizon
UV (~350 nm) 440/40 NA Alexa Fluor 350 LIVE/DEAD DAPI Hoechst 33342 FxCycle Violet Pacific Blue Alexa Fluor ECFP BUV737
450/50 Alexa Fluor 405 Fixable Blue SYTOX Blue (DAPI) 350
Vybrant DyeCycle BD Horizon
Violet BV421

740/35 NA BD Horizon
V450
425/20 VL1 440/50 Super Bright 436 LIVE/DEAD DAPI Vybrant DyeCycle FxCycle Violet Annexin V, CellTrace Violet Azurite
440/50 eFluor 450 Fixable Violet SYTOX Blue Violet Pacific Blue Cerulean VioBlue
450/50 V L1( V6 * * ) Pacific Blue Click-iT Plus EdU TagBFP
455/50 450/40 Annexin V, Pacific Blue BD Horizon
450/65 Alexa Fluor 405 eFluor 450 ECFP BV510
460/50 mTurquoise
PO-PRO-1 BD Horizon
AmCyan V500
510/50 VL2 512/25 eFluor 506 LIVE/DEAD Violet
520/35 VL2(V6) 525/50 Pacific Green Fixable Aqua Ratiometric T-Sapphire VioGreen
525/50 BD Horizon
Violet (405 nm) 530/40 Probe
550/40 (F2N12S) BV570
BD Horizon
585/40 VL3 603/48 Super Bright 600 LIVE/DEAD Violet
610/20 VL3(V6) 610/20 Pacific Orange Fixable Yellow Ratiometric BV605
BD Horizon
Qdot 605 Probe
(F2N12S) BV650
BD Horizon
660/20 VL4 710/50 Super Bright 645
660/40 VL4(V6) 660/20 Super Bright 702 BV650
710/50 BD Horizon
710/50 Qdot 705
780/60 BV711
VL5 710/50 Super Bright 702 BD Horizon
525/50
525/30 VL6 780/60 Super Bright 780 BV711
525/40 BD Horizon
530/30 BL1 530/30 Alexa Fluor 488 LIVE/DEAD SYTOX Green Vybrant DyeCycle Annexin V, CellTrace CFSE CellROX Green pHrodo Green EGFP
530/40 BL1(G†) 525/50 FITC Fixable Green Green Alexa Fluor E. coli Emerald GFP BV786
7-A A D Click-iT Plus EdU
574/26 LIVE/DEAD Propidium Iodide Vybrant DyeCycle 488 Alexa Fluor BioParticles EYFP BD Horizon
575/30 Fixable Red Orange 488 Conjugate BB515
580/23 (PI) Annexin V, FITC
585/40 SYTOX pHrodo Green BD Horizon
586/15 AADvanced APO-BrdU S. aureus PE-CF594
590/40 SYTOX Orange TUNEL with
670/30 Alexa Fluor BioParticles PE/Dazzle 594
675/30 Conjugate
680/30 488 PerCP-Vio700
Blue (488 nm) 695/30
695/40 CellEvent PE-Vio770
710/50 Caspase-3/7
750 LP
775/50 Green
780/40
780/60 MitoProbe
DiOC2(3)

MitoProbe JC-1

YO-PRO-1
Iodide

BL2 574/26 PE FxCycle PI/RNase Annexin V, PE pHrodo Red E. coli EYFP
PE-eFluor 610 TMRE BioParticles mCitrine
BL2(G,Y‡) PE-Alexa Fluor 610 TMRM Conjugate Venus
590/40 PE-Texas Red
MitoProbe JC-1 pHrodo Red
Phagocytosis

Kit

BL3 695/40 PerCP-eFluor 710 7-A A D Annexin V,
BL4 780/60 PE-Alexa Fluor 700 PerCP-eFluor
Propidium Iodide
PE-Cy®5.5 710
PerCP SYTOX
AADvanced
PerCP-Cy®5.5
Qdot 705 Vybrant DyeCycle Annexin V,
TRI-COLOR Ruby PerCP-eFluor
PE-Cy®5)
710
PE-Cy®7
Qdot 800

12

Excitation Common Attune NxT Recommended Viability dyes Viability dyes DNA content/ DNA content/ Apoptosis Cell ROS Phagocytosis Fluorescent Other dyes
laser emission channel dyes (compatible (non-fixed cell cycle cell cycle dyes proliferation detection dyes proteins
filters (nm) (nm)* with fixation) cells) dyes (live dyes (fixed DsRed PE/Dazzle 594
GL1 575/36 Alexa Fluor 532 cells) cells) Annexin V, R-PE dyes dyes tdTomato
555/20 Alexa Fluor 555 LIVE/DEAD SYTOX Orange YFP ECD
575/24 GL2 620/15 Fixable Red Vybrant DyeCycle FxCycle PI/RNase Annexin V, CellTrace Yellow pHrodo Red E. coli BD Horizon
585/16 GL3 695/40 Qdot 565 SYTOX Orange Alexa Fluor mCherry
585/42 Qdot 605 AADvanced Click-iT Plus EdU CellROX Orange BioParticles PE-CF594
610/20 R-phycoerythrin Propidium Iodide Vybrant DyeCycle 555 Alexa Fluor mOrange PE/Dazzle 594
610/20 (R-PE, PE) Orange 594 Conjugate RFP
620/15 MitoTracker PE-Vio 615
Green (532 nm) 630/30 Alexa Fluor 568 Orange DsRed
Alexa Fluor 594 mCherry
695/40 CMTMRos mKate
710/50 Qdot 605 mStrawberry
PE-Texas Red Annexin V, tdTomato
Alexa Fluor
Qdot 705
PE-Alexa Fluor 700 568

TRI-COLOR Annexin V,
PE-Cy®5.5 Alexa Fluor

594

MitoTracker
Red CMXRos

SYTOX Vybrant DyeCycle
AADvanced Ruby

780/60 GL4 Qdot 800
780/60 PE-Cy®7

585/40 YL1 Alexa Fluor 555 Propidium Iodide Vybrant DyeCycle MitoTracker pHrodo Red E. coli
585/15 585/16 PE Orange
590/40 SYTOX Orange Orange CellTrace Yellow CellROX Orange BioParticles
CMTMRos
Conjugate
MitoTracker
Red CMXRos

TMRE

TMRM

Yellow (561 nm) 610/20 YL2 620/15 PE-eFluor 610 LIVE/DEAD 7-A A D Vybrant DyeCycle FxCycle PI/RNase Annexin V, Click-iT Plus EdU
613/20 PE-Alexa Fluor 610 Fixable Red Orange Alexa Fluor Alexa Fluor
615/50 Propidium Iodide 594
620/15 PE-Texas Red 568
620/30 SYTOX
AADvanced Annexin V,
Alexa Fluor
SYTOX Orange
594

670/30 YL3 695/40 PE-Alexa Fluor 700 7-A A D mPlum
675/30 PE-Cy®5.5 mRaspberry
680/30 Y L 3( B §/ Y/ R ††/ Qdot 705 Propidium Iodide mNeptune
695/30 V6) TRI-COLOR
695/40 SYTOX
780/60 AADvanced

750 LP YL4 780/60 PE-Cy®7 Vybrant DyeCycle PE-Vio770
780/40 Qdot 800 Ruby
780/60 BD APC-H7
660/20 RL1 670/14 Alexa Fluor 647 LIVE/DEAD SYTOX Red FxCycle Far Red Annexin V, CellTrace Far Red CellROX Deep APC-Vio770
661/20 eFluor 660 Fixable Far Red Alexa Fluor Red
665/20 APC Click-iT Plus EdU
670/14 Qdot 655 647 Alexa Fluor
670/30 APC-Cy®5 647
675/30 Annexin V,
Red (637 nm) Alexa Fluor
720/30
730/45 680
750 LP
775/50 Annexin V, APC
780/40
780/60 MitoProbe
DiIC1(5)

TO-PRO-3

RL2 720/30 Alexa Fluor 680 LIVE/DEAD Vybrant DyeCycle
RL3 780/60 Alexa Fluor 700 Fixable Near-IR Ruby
APC-Alexa Fluor

700
Qdot 705

APC-eFluor 780

APC-Alexa Fluor
750

APC-Cy®7

* Filter sets change depending on the configuration of the Attune NxT Flow Cytometer in use.
** V6 is the Attune NxT violet 6-channel configuration option.
† G refers to the green laser option.
‡ Y refers to the yellow laser option.
§ B refers to the blue laser option.
†† R refers to the red laser option.

13

Beads for instrument controls and standards

Compensation beads Measured data Analysis results
For proper interpretation of collected data, it is important
to know that the fluorescence signal being recorded for Figure 9. Spectral overlap is eliminated by compensation.
your dye of choice comes from that particular dye, and
not from another dye that happens to emit some light Mouse IgG1, k (RPA-T4)
in the same wavelength range. Capturing the correct Mouse IgG2a, k (OKT3)
emission data is aided by proper compensation (Figure 9). Mouse IgG2b, k (OKT4)
Invitrogen™ eBioscience™ UltraComp eBeads compensation Mouse IgG3, k (G4.18)
beads provide options to suit your flow cytometry antibody Mouse IgG1, k (RPA-T4)
compensation needs (Table 2 and Figure 10). The Mouse IgM, k (HI98)
Invitrogen™ ArC Amine Reactive Compensation Bead Kit is Rat IgG1, k (PC61.5)
available for compensating the LIVE/DEAD Fixable Dead Rat IgG2a, k (RM4-5)
Cell Stains and the Invitrogen™ GFP BrightComp eBeads Rat IgG2b, k (30-F11)
Compensation Beads provide a consistent, accurate, Rat IgG2c, k (49-H4)
and simple-to-use reagent for setting flow cytometry Rat IgGM, k (DX5)
compensation when using green fluorescent protein (GFP) Rat IgG, lambda (93)
(Table 2). Golden Syrian Hamster IgG (500A2)
Armenian Hamster IgG (145-2C11)
Compensation beads are suitable for the
following scenarios: Figure 10. Staining of UltraComp eBeads with 13 different eFluor 450
dye–conjugated monoclonal antibodies including one of each
• Sample cells are in limited supply for subclass commonly used in flow cytometry. Beads were stained with
making compensation 0.25 μg of each antibody and analyzed by flow cytometry. Each histogram
represents one staining antibody (clone and isotype indicated at right).
• Signal compensating of a positive population is needed

• Performing experiments where the panels use
fluorophores whose emissions patterns overlap

Learn more at thermofisher.com/flow-compensation

Table 2. Invitrogen™ and eBioscience™ flow cytometry compensation bead products.

UltraComp eBeads GFP BrightComp eBeads™ ArC™ Amine Reactive Compensation
Compensation Beads Bead Kit

Reactivity Hamster, mouse, and rat antibodies, Near-identical, spectral match to GFP LIVE/DEAD™ fixable dead cell stains
and recognition of the kappa and
lambda chains

Format One vial, dispense as a single drop 1 vial positive beads
1 vial negative beads

Laser Compatible with most standard lasers, Designed for use with 488 nm Compatible with most standard lasers,
compatibility UV to 633 nm (blue) lasers UV to 633 nm

14

Calibration and size reference beads Cell counting beads
Instrument calibration is critical to collecting and analyzing Absolute cell counts have been widely used in quantifying
accurate experimental data. Our flow cytometer alignment cell concentration or absolute count of cells in a
and cell sorting beads are designed to help ensure your sample. We offer two cell counting products: Invitrogen™
flow cytometer is performing at its peak, your experimental CountBright™ Absolute Counting Beads and AccuCheck™
design is robust, and the data you collect and analyze Counting Beads (Figure 11). They are both designed to
are accurate. In addition, our size calibration and size work on any sample type, and the AccuCheck beads also
reference kits serve as reliable size references for flow provide a pipetting accuracy check.
cytometer users.
Find out more at
• Confidence—ensure the reliability of optimal daily thermofisher.com/flow-cell-counting-beads
instrument performance

• Reliability—minimal variation for consistent
data acquisition

• Compatibility—full range of tools for any instrument

For more information, go to
thermofisher.com/flowcalibration

Figure 11. CountBright Absolute Counting Beads. A mixture of live
and heat-killed Jurkat cells were treated with reagents in the LIVE/DEAD
Viability/Cytotoxicity Kit. CountBright Absolute Counting Beads were
added to the sample, which was then analyzed by flow cytometry using
488 nm excitation. Calcein fluorescence was collected with a 530/30 nm
bandpass filter and ethidium homodimer-1 (EthD-1) fluorescence was
collected with a 610 nm longpass filter. The data show clear separation of
live and dead cells, as well as separation of the counting beads.

15

Sample preparation buffers and reagents

The selection of fixation and permeabilization buffer Secreted protein: cytokine, chemokine, and
systems has a significant impact on the quality and growth factors
accuracy of the data when performing intracellular Cytokines, chemokines, and growth factors can pose
staining for flow cytometric analysis. It is important to additional difficulties during evaluation by flow cytometry.
consider the location of the target proteins within a cell to These proteins go through a secretory pathway; and as
select the appropriate buffer. We provide buffer sets that such, they require protein accumulation in addition to
are optimized for detection of nuclear proteins such as fixation and permeabilization for optimal detection in flow
transcription factors, as well as cytoplasmic and secreted cytometry analysis. This can be accomplished by blocking
proteins. To find out more about choosing the appropriate protein movement to different organelles and secretion
buffer system, go to thermofisher.com/flow-sample. through the use of transport inhibitors. This is often
achieved with brefeldin A, which blocks transport at the
Cytoplasmic staining endoplasmic reticulum; or with monensin, which blocks
Cytoplasmic staining requires fixation to crosslink transport at the Golgi apparatus. Invitrogen™ eBioscience™
proteins and stabilize the cell membrane, followed by Cell Stimulation Cocktails contain the stimulants PMA
permeabilization to allow antibodies access to intracellular and ionomycin, with or without the inclusion of protein
antigens, thereby preventing cell contents from escaping transport (secretory pathway) inhibitors at a ready-to-use
once the cell has been permeabilized. Intracellular fixation concentration (Figure 12).
and permeabilization buffers are ideal for optimal detection
of cytoplasmic proteins, including cytokines and other
secreted proteins. The Invitrogen™ FIX & PERM™ Cell
Permeabilization Kit is designed for use when staining
proteins, such as adaptor proteins ß-catenin, actin,
and tubulin, in addition to receptor proteins in which
the antibody recognizes a cytoplasmic version (CD152
(CTLA-4)) or cytoplasmic domain.

IL-17A FITC
IL-17A PE
IL-22 PerCP-eFluor 710
IL-22 PerCP-eFluor 710

CD4 eFluor 450 IL-21 eFluor 660 IL-17PE IL-21 eFluor 660

Figure 12. Human Th17 cytokine staining panel in CD4+ T cells. Th17-polarized CD4+ PBMCs were stimulated for 5 hours with Cell Stimulation Cocktail
(plus protein transport inhibitors) and then intracellularly stained with the Invitrogen™ eBioscience™ Human Th17 Cytokine Staining Panel. Lymphocytes
were gated for staining of CD4 eFluor 450 dye and IL-17A FITC (left plot, blue) and then analyzed for staining of IL-17F PE, IL-21 eFluor 660, and IL-22
PerCP–eFluor 710.

16

Nuclear staining Blocking Fc-mediated nonspecific antibody binding
Transcription factors are DNA-binding proteins that regulate High background reduces data quality, making
gene expression. Detection of transcription factors in analysis difficult; but by using human, canine, or rhesus
populations at low frequencies can be challenging if the Fc receptor–blocking reagents or an anti-mouse
appropriate buffers are not utilized. Fortunately, the Foxp3/ CD16/32 antibody, optimal staining and signal-to-noise
Transcription Factor Staining Buffer Set (although originally ratios can be achieved. Fc-blocking reagents are used to
developed for Foxp3 staining) has been optimized for use inhibit unwanted and nonspecific binding of antibodies to
with nuclear factors, cytosolic proteins, secreted proteins, Fc receptor (FcγR), thereby allowing optimal staining and
and transcription factors, in addition to cytokines. These signal-to-noise ratios during flow cytometric analysis. To
include Eomes, T-bet, Gata-3, IRF4, phospho-H2Ax, find the blocking reagent or antibody that you need, go to
Rorγ(t), Egr2, Ki-67, and Sox2 (Figure 13). Additionally, this thermofisher.com/flowantibodies.
buffer has been proven to work successfully when co-
staining with many secreted proteins such as IL-17A and
granzyme B.

IL-17A APC
IL-17A APC
% of max

CD4 PE-Cyanine5.5 CD4 PE-Cyanine5.5 RORγ(t) PE

Figure 13. Identification of Th17 cells by flow cytometric detection of RORγ(t). CD4+ T cells were sorted from RORγ(t)-deficient (left plot) or wild-
type (middle plot) mouse spleen and lymph nodes, cultured in Th17-polarizing conditions for 3 days, and stained with Invitrogen™ Anti–Mouse CD4 PE-

Cyanine5.5, Anti–Mouse IL-17A APC, and Anti–Human/Mouse RORγ(t) PE. The histogram shows staining of RORγ(t) in CD4+/IL-17A+–gated events from
RORγ(t)-deficient mice (blue line) and wild-type mice (pink line) (right plot). Cells in the lymphocyte gate were used for analysis. Data provided courtesy of
Dr. Littman, New York University.

17

Instrumentation

Attune NxT Flow Cytometer and Autosampler

Speed isn’t just about going fast; it’s about “We have yet to clog the machine with our debris-rich
enabling discovery primary tumor samples. Of course, the acoustic
Run samples faster and achieve greater resolution—with technology greatly facilitates the identification of small
little fear of sample loss due to clogging. The Attune NxT populations, like cancer stem cells, increasing our
Flow Cytometer with Autosampler combines precision capacity to detect and quantify these rare events with
with performance in a benchtop flow cytometer that is high efficiency and reliability.”
configurable with up to four lasers and 16 parameters of
detection (Table 3). Do more. Find more. Explore more. – Bruno Sainz Jr, PhD, Autonoma University
of Madrid, School of Medicine
• Transform your research—get a superior level of data
fidelity at speeds up to 1 mL/min; discover rare cells and “I knew I had learned a lot during 25 years of experience
analyze more cells in a shorter period of time doing research with flow cytometry. Now I am surprised to
see how much I can learn doing research with the Attune
• Six fluorescence channels off the violet laser— NxT (instrument), and how this new technology can be very
expand your capabilities in multicolor flow cytometry helpful to make visible the invisible.”

• Enable new applications and sample types—high – Dr. Jordi Petriz, PI at Josep Carreras Leukaemia Research
sample processing rates make it possible to evaluate Institute, Barcelona, Spain; executive board member at the
difficult sample types (such as tumors) that you may not European Society for Clinical Cell Analysis (ESCCA)
have been able to investigate before
“We looked at several metrics and compared the
• Expand your capabilities—system modularity with up Attune NxT Autosampler to other 96-well plate readers.
to four lasers and 16 detection channels allows you to The Autosampler proved to have very good stability
upgrade as your research needs expanding and very low carryover. We were most impressed
by the way that the autosampler took advantage of
• Simplified sample prep—no-wash, no-lyse sample prep the Attune NxT Flow Cytometer’s fluidics and high-volume
options streamline your workflow throughput. Without compromising stability or
precision, the Autosampler was able to run plates much
• Flexibility—convert between tubes and plates with a faster than any other plate reader.”
simple click of the mouse
– E. M. Myer, University of
• Option for automation—designed for walk-away Pittsburgh Cancer Institute
performance with clog-resistant fluidics and robust data
analysis software

• Run reliably for hours at a time—The Attune NxT
instrument is available with robotic automation.

• Microplate taxiing—automated with extended
unmanned runtime settings, scalable configuration
flexibility, and maximized capacity for processing
staggering quantities of data

Learn more at thermofisher.com/attune

18

Table 3. Laser configurations and antibody options for the Attune NxT Flow Cytometer.

Excitation Channel Antibody labels Blue Blue/ Blue/ Blue/ Blue/ Blue/ Blue/ Blue/ Blue/ Blue/ Blue/ Blue/ Blue/ Blue/ Blue/
laser Super Bright 436 violet violet 6 green red yellow red/ red/ violet/ violet/ red/ red/ red/ red/ red/
VL1 (440/50) Alexa Fluor 405 violet violet 6 yellow green green yellow yellow/ yellow/ violet/
(450/40)* eFluor 450 • •* • • violet violet 6 green
Pacific Blue •* •
VL2 (512/25) eFluor 506 • •* • • •* • • • •* •
(525/50)* Pacific Green • •* • •
Violet (405 nm) VL3 (603/48) Super Bright 600 • •* • • •* • • • •* •
(610/20)* Pacific Orange • •
VL4 (710/50) • • • • •* • • • •* •
(660/20)* Super Bright 645 • • •* • • • •* •

VL5 (710/50) Super Bright 702 • •
• •
VL6 (780/60) Super Bright 780 • • • •* •* • • • •*

BL1 FITC • • • •*
(530/30)
(525/50)* Alexa Fluor 488

BL2 PE
(590/40)
(574/26)* PE-Alexa Fluor •* •* •* •* •* • • •* • (695/40) •
610

Blue (488 nm) PE-Texas Red

PE-Alexa Fluor
700

BL3 PE-Cy5 •••• •••• •• •
(695/40) PE-Cy5.5
PerCP-Cy5.5

PerCP-eFluor
710

BL4 PE-Alexa Fluor • • ••
(780/60) 700
PE-Cy7

GL1 Alexa Fluor 532 • •• •
(575/36) Alexa Fluor 555
PE

Green (532 nm) GL2 PE-eFluor 568 • •• •
(620/15) Alexa Fluor 594
PE-Texas Red

GL3 PE-Alexa Fluor • •• •
(695/40) 700
PE-Cy5.5

GL4 PE-Cy7 • •• •
(780/60)

YL1 (585/16) PE • ••••

Yellow (561 nm) YL2 (620/15) PE-eFluor 610 • ••••
YL3 Alexa Fluor 568
(695/40) PE-Texas Red • ••••
YL4 PE-Alexa Fluor • ••••
(780/60) 700 •• ••••
RL1 (670/14) PE-Cy5
Red (637 nm) RL2 PE-Cy5.5 •• ••••
(720/30)
PE-Cy7 •• • ••••
RL3
(780/60) APC
eFluor 660
Alexa Fluor 647
Alexa Fluor 680
Alexa Fluor 700
APC-Alexa Fluor
700
Alexa Fluor 750
APC-Alexa Fluor
750
APC-eFluor 780
APC-Cy7

* Denotes Attune NxT Flow Cytometer Violet 6–channel option

19

Resources

Reference guides Stay in the know—news and updates

The Molecular Probes™ Handbook, 11th Edition BioProbes™ Journal
The most complete reference on fluorescent Our award-winning print and online magazine,
labeling and detection available, this resource BioProbes Journal, highlights the latest
features extensive references and technical notes breakthroughs from our scientists, featuring new
and contains over 3,000 technology solutions technologies and products.
representing a wide range of biomolecular labeling
and detection reagents. See the online version of To access current and past issues of the BioProbes
The Molecular Probes Handbook and request your Journal, go to thermofisher.com/bioprobes
free copy* at thermofisher.com/handbook
Service and support
Online tools
We offer answers to frequently asked questions, and extensive
Flow cytometry antibody selection tool troubleshooting guides for flow cytometry experiments. To browse
Explore our extensive portfolio of high- recommendations from our professionals or to contact a technical support
quality primary and secondary antibody representative, go to thermofisher.com/flow-support
conjugates with this easy-to-use selection tool.
thermofisher.com/flowantibodies

Learn more

Invitrogen™ Flow Cytrometry Panel Builder Flow Cytometry Learning Center
This online tool guides you through flow cytometry Search for protocols, tutorials, application notes,
panel design, providing a simplified, customizable fluorophore and product selection guides, literature,
experience to fit your flow cytometry panel design and many other technical resources in a single place.
needs. thermofisher.com/flowpanel thermofisher.com/flowlearning

Interactive instrument 3D explorer demo @facebook.com/thermofisher
Explore the features of the Attune NxT Flow @facebook.com/invitrogen
Cytometer with this virtual demo. Spin the @facebook.com/molecularprobes
unit around and interact with key features. @twitter.com/servingscience
thermofisher.com/attune3ddemo @twitter.com/invitrogen
@twitter.com/molprobes
JUNE 2015 BioProbes 71
Our award-winning print and digital magazine @linkedin.com/thermo-fisher-scientific
BIOPROBES 71 highlights the latest breakthroughs in cell biology,
featuring new technologies and products. Read the
MOLECULAR PROBES™ JOURNAL OF CELL BIOLOGY APPLICATIONS latest issue at thermofisher.com/bioprobes
SPECIAL FLOW CYTOMETRY ISSUE

A sensitive flow cytometry assay
for hyperploidy and cell death
in the Drosophila brain

PLUS
Flow cytometry panel design
Tools and strategies for rare-event detection
Multiparameter detection of early apoptosis markers
Instrument specifications for the Attune™ NxT cytometer

* Not available in all countries.

Find out more at thermofisher.com/flow

For Research Use Only. Not for use in diagnostic procedures. Not for resale. Super Bright Polymer Dyes are sold under
license from Becton, Dickinson and Company. © 2019 Thermo Fisher Scientific Inc. All rights reserved. Cy is a registered
trademark of GE Healthcare. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise
specified. COL09364 0419

Molecular Probes flow cytometry

Products and resource guide

Molecular Probes flow cytometry

Products and resource guide

Flow cytometry workflow Attune NxT flow cytometer
The Attune NxT Flow Cytometer is a benchtop analyzer that
Reagents uses a revolutionary technology—acoustic focusing—to align
The Invitrogen™ Molecular Probes™ flow cytometry products cells or particles prior to the laser interrogation points. Now
and resource guide presents an overview of primary antibody with the flexibility to create a customized 4-laser, 14-color
conjugates, cell function assays, and other tools optimized for system, the Attune NxT flow cytometer is designed to
flow cytometry by our scientists. We have been at the forefront accommodate a variety of experimental protocols and any
of invention and development of fluorescent probes for over lab budget.
40 years, and this guide features some of the most useful flow
cytometry tools available anywhere. For the probes and assays See page 32 for more information.
you need, available in a full spectrum of fluorescent colors, look
to Molecular Probes flow cytometry products first.

Antigen detection 12
Primary antibodies 12
Fluorophore overview 15
Antibody labeling 18
Secondary detection 19
Custom services

Sample preparation Instrument set-up
Cell preservation
Red blood cell lysis 2 and calibration
Fixation and permeabilization 2
Dynabeads™ cell isolation 2 Alignment 6
3
Size calibration 6

Cell sorting set-up 7 Cell analysis

Compensation 7 Cell viability 20

Absolute cell counting 10 Cell proliferation 22

Cell cycle 24

Apoptosis 25

Other cell function assays 28

Visit thermofisher.com/flow-cytometry for more information on Molecular Probes flow cytometry products, the Attune NxT
flow cytometer and flow cytometry resources.

1

Sample preparation

Sample preparation Quality data require quality starting material. Invitrogen™ Fixative-free lysing solution High-Yield Lyse
Molecular Probes™ sample preparation reagents, which include Invitrogen™ High-Yield Lyse (Cat. No. HYL250) is a premixed,
reagents for blood cell preservation, red blood cell lysis, and fixative-free erythrocyte lysing solution that can be used
sample fixation and permeabilization, are designed to help to eliminate red cells from whole blood for flow cytometric
you achieve the best possible results. Learn more about these analysis, with minimal loss of rare blood cell populations.
products and get protocols at thermofisher.com/flow-sample Using this reagent, erythrocyte lysis is performed immediately
following staining of the blood samples with monoclonal
Cell preservation antibodies; therefore, there is no need for a wash step.

TransFix Reagent Fixation and permeabilization
Invitrogen™ TransFix™ Cellular Antigen Stabilizing Reagent (Cat.
No. FIX2, FIX20, FIX100) is a whole-blood preservative that FIX & PERM reagents and kits
stabilizes leukocytes and preserves antigenic sites for future • Compatible with analysis of most cellular antigens
cellular analysis. The reagent allows simple batch processing
of multiple samples to optimize your laboratory workflow. • No effect on cellular morphological scatter
Additionally, blood that has been stabilized with TransFix
Reagent can be easily transported to other sites for analysis. • Reduced background staining

• Proven protocols

Cell lysis The Invitrogen™ FIX & PERM™ Cell Permeabilization Kit (Cat.

Cal-Lyse whole blood lysing solution No. GAS003, GAS004) consists of matched Fixation Medium
The Invitrogen™ Cal-Lyse™ premixed lysing solution (Cat. No.
GAS010, GAS010S100) is specifically formulated to lyse red (Medium A) and Permeabilization Medium (Medium B) for
blood cells following monoclonal antibody staining of whole
blood or bone marrow. Treatment with this reagent leads to simultaneous analysis of intracellular and cell-surface antigens
both the lysis of red blood cells and the fixation of white cells.
Treatment does not affect fluorophore-labeled antibodies in the same cell population (Figure 1). This procedure facilitates
bound to leukocytes, and leaves morphological light-scattering
characteristics of leukocytes intact. Cal-Lyse reagent can be antibody access to intracellular structures and leaves the
used for either “no-wash” or “wash” staining procedures.
TNF-α–Paci c Blue™ uorescencemorphological light-scattering characteristics of the cellsTNF-α–Paci c Blue™ uorescence
intac10t6 . These formulations reduce106 background staining and

105 105

allow simultaneous addition of permeabilization medium and

fluorophore-labeled antibodies. The Fixation Medium (Cat.

No.10G4 AS001S100) and Permeab10i4lization Medium (Cat. No.

GAS1003 02S100) are available sepa10r3ately as well.
-103 -103

-103 103 104 105 106 -103 103 104 105 106

γ-IFN–PE-Cy®7 uorescence γ-IFN–PE-Cy®7 uorescence

A TNF-α–Paci c Blue™ uorescence TNF-α–Paci c Blue™ uorescence 106 BTNF-α–Paci c Blue™ uorescence γ-IFN–PE-Cy®7 uorescence 106
105
106 106

105 105 105

104 104 104 104

103 103 104 105 106 103 103 104 105 106 103 103 104 105 106 103 103 104 105 106
-103 -103 -103 -103
CD4– uorescein uorescence CD4– uorescein uorescence
-103 -103 -103 -103

γ-IFN–PE-Cy®7 uorescence γ-IFN–PE-Cy®7 uorescence

TNF-α–Paci c Blue™ uorescenceFigure 1. U1s06e of FIX & PERM Cell Permeabili1z0a6 tion Kit for simultaneous surface antigen and intracellular antigen staining. C57BL/6 splenocytes were leftγ-IFN–PE-Cy®7 uorescence
unstimulated or stimulated for 5 hours with phorbol myristate acetate (PMA) and ionomycin in the presence of brefeldin A. Cells were then surface-stained with
fluorescein1(0F5 ITC)-conjugated anti-mouse CD4 a10n5 tibody (Cat. No. MCD0401). This step was followed by fixation and permeabilization of the sample using the FIX

& PERM Cell Permeabilization Kit (Cat. No. GAS003, GAS004). Intracellular staining was performed during the permeabilization step using Invitrogen™ PE-Cy®7

tandem–conjugated anti-mouse γ-interferon (γ-IFN) antibody (Cat. No. A18713) and Invitrogen™ Pacific Blue™ dye–conjugated anti-mouse tumor necrosis factor α
(TNF-α) ant1i0b4 ody (Cat. No. RM90128). Data wer1e04 collected using the Attune flow cytometer (blue/violet) with 488 nm excitation and a 530/30 nm bandpass emission
filter to detect FITC fluorescence and a 640 nm longpass filter to detect PE-Cy7 tandem fluorescence. Pacific Blue conjugate fluorescence was detected using 405

nm excitatio10n3 and a 450/40 nm bandpass emiss10i3on filter. (A) γ-IFN and TNF-α antibody co-staining of total mouse splenocytes, gated on lymphocytes, that were
left unstimu-10la3-1t0e3 d10(3left) or st1im04 ulate10d5 (r1i0g6 ht) with-1P03M-10A3 1a03nd ionom1y04cin in10t5he10p6 resence of brefeldin A. (B) CD4+ T cell expression of TNF-α (left) and γ-IFN (right) after
stimulation as deCsDc4r–ibueodresacbeionveuo.rescence
CD4– uorescein uorescence

2

Dynabeads cell isolation Figure 2. Dynabeads products are uniform
spherical beads with highly defined and
• Viable and functional—products for positive isolation, negative isolation, cell consistent product characteristics, which helps
activation, and depletion ensure that you get truly reliable and reproducible
results.
• Gentle—column-free separation and inert bead surfaces translate to gentler
handling of your cells and help reduce the risk of contaminants in the preparations Incubate your
starting sample
• High yields—tube-based separation allows you to achieve excellent recovery with Dynabeads™
of cells product

When cells are removed from their natural environment, there is a risk that Separate
experimental procedures will negatively impact cell phenotype and function. bead-captured
Choosing the right cell separation method is therefore critical to downstream cells with a
experiments. Invitrogen™ Dynabeads™ magnetic beads are superparamagnetic, Dynal MPC™
monosized polymer beads coated with a thin, inert polymer shell to encase magnet
the magnetic material (Figure 2). This design helps to reduce the risk that any
unwanted material such as iron is left in the sample after separation. In addition,
since the cells are never bound to the beads (negative-isolation and depletion
beads) or are released from the beads after the gentle magnetic separation
(positive-isolation beads), the final cell sample is of high purity and viability, with
no process-derived remnants that could affect the results (Figure 3).

Human cell isolation Transfer
Dynabeads magnetic beads enable gentle tube-based isolation of human cells supernatant with
directly from whole blood, mononuclear cells, buffy coat, bone marrow, or untouched cells
tissue samples for any downstream assay, including flow cytometry (Figure 4). to a new tube
Dynabeads products are available for isolating human T cells, B cells, stem cells,
NK cells, monocytes, dendritic cells, endothelial cells, tumor cells, leukocytes, and Negatively Positively
granulocytes. isolated cells isolated cells
(bead-captured)
Isolation options include: (untouched)

• Positive isolation and cell release 1

• Negative isolation, resulting in untouched cells 2 beads
Remove
• Depletion of unwanted cell types or positive cell isolation for 3
molecular applications
Cell-based assays, flow Molecular applications
• Isolation of cells using your own antibody cytometry, cell expansion e.g., mRNA and DNA analysis

If you can’t find a ready-to-use product for human cell isolation, we have a range Figure 3. Workflow diagram for using
of Dynabeads products that can be combined with an antibody of your choice Dynabeads beads for positive or negative tube-
to create a tailored cell isolation tool that include: streptavidin beads, secondary based cell isolation.
antibody–coated beads, and surface-activated beads. Learn more at
thermofisher.com/humancellisolation 1,000 1,250 2,000
1,000 1,250 2,000

Count 1,500
500Count750 1,500
500 750
Count
1C,o0u0n0t
1,000

250 500
250 500

0 0
0 0

102102 103103 104104 105105 102102 103103 104104 105105

CDC4DF4ITFCITC CDC4DF4ITFCITC

Figure 4. Purity of human CD4+ T cells. Purity
before (left) and after (right) negative isolation
from peripheral blood mononuclear cells using the

Invitrogen™ Dynabeads™ Untouched™ Human CD4

T Cells Kit (Cat. No. 11352D).

3

Sample preparation Mouse cell isolation Find more information online
Dynabeads products offer gentle tube-based isolation of If you go to thermofisher.com/cellisolation, you can find
mouse cells directly from whole blood, spleen, lymph node, or information about our entire line of cell isolation products, and
thymus for any downstream assay, including flow cytometry you’ll also have access to:
(Figure 5). Dynabeads products are available for isolating
mouse T cells, B cells, NK cells, and dendritic cells. • Selection guides for choosing the correct cell isolation
product
Isolation options include:
• Protocols for sample preparation and strategies for cell
• Positive isolation and cell release isolation

• Negative isolation of untouched cells • Data showing the performance of our cell isolation
products versus other commercially available products
• Depletion of unwanted cell types or positive cell isolation
for molecular applications • Help in choosing the correct magnets for your tubes or
plates
If you can’t find a ready-to-use product for mouse cell
isolation, we have a range of Dynabeads products that can • Links to videos, brochures, and application notes and to
be combined with an antibody of your choice to create a references that cite the use of Dynabeads products
tailored cell isolation tool: streptavidin beads, secondary
antibody–coated beads, and surface activated beads.
Learn more at thermofisher.com/mousecellisolation

Dynabeads™ FlowComp™ Mouse CD4 Kit

600 Purity: 105
500 97.2%
400
300 Propidium iodide 104
200
Count 100 103

0 102 Viability:
86%
400
350 102 103 104 105 102 103 104 105
300 CD4+ CD4+
250
200 Column-based isolation
150
100 Purity: 105
78.5%
50
0 Propidium iodide 104

Count 103

102 Viability:
63%

102 103 104 105 102 103 104 105
CD4+ CD4+

Figure 5. Isolation of CD4+ T cells from mouse spleen cells. Cell isolation using the Invitrogen™ Dynabeads™ FlowComp™ Mouse CD4 Kit (Cat. No. 11461D)
(top) results in substantially higher purity (97%) and viability (86%) than column-based (bottom) positive cell isolation (yielding purity and viability of 78% and 63%,
respectively).

4

Sample preparation product list

Section name Product name Species Target cell Regulatory Size Cat. No.
status
NA FIX2
Research Use Only (RUO) FIX20
T cells FIX100
Cell preservation TransFix Cellular Antigen Stabilizing Reagent NA T cells RUO* 2 mL 11361D
T cells 20 mL 11331D
Dynabeads cell Dynabeads FlowComp Human CD4 Kit Human T cells RUO 100 mL 11352D
isolation Dynabeads CD4 Positive Isolation Kit Human T cells RUO 3 mL 11362D
Human T cells RUO 5 mL 11333D
Dynabeads Untouched Human CD4 T Cells Kit Human T cells RUO 2 mL 11348D
Human T cells RUO 3 mL 11461D
Dynabeads FlowComp Human CD8 Kit Human T cells RUO 5 mL 12406D
Mouse T cells RUO 1 kit 11415D
Dynabeads CD8 Positive Isolation Kit Mouse T cells RUO 3 mL 11462D
Mouse RUO 5 mL 11417D
Dynabeads Untouched Human CD8 T Cells Kit Mouse NA RUO 2 x 10 mL
Mouse RUO 3 mL GAS003
Dynabeads FlowComp Mouse CD4 Kit NA 2 x 10 mL GAS004
NA NA GAS001S100
DETACHaBEAD Mouse CD4 Kit GAS002S100
NA NA
Dynabeads Untouched Mouse CD4 Cells Kit NA GAS010
NA GAS010S100
Dynabeads FlowComp Mouse CD8 Kit NA HYL250

Dynabeads Untouched Mouse CD8 Cells Kit

General Purpose Reagent (GPR)

Fixation and FIX & PERM Cell Permeabilization Kit GPR** 50 assays
permeabilization 200 assays
GPR 100 mL
Fixation Medium (Medium A) GPR 100 mL
Permeabilization Medium (Medium B)
In Vitro Diagnostic (IVD) IVD† 25 mL
Red blood cell lysis Cal-Lyse Whole Blood Lysing Solution IVD 100 mL
500 mL
High-Yield Lyse NA

NA, not applicable.
*RUO: For Research Use Only. Not for use in diagnostic procedures, unless otherwise indicated.
**GPR: For Laboratory Use.
†IVD: For In Vitro Diagnostic Use.

5

Instrument set-up and calibration

Set-up and calibration • Helps to ensure the reliability of optimal daily instrument determined by transmission electron microscopy. The size
performance of cells in an experimental sample can be estimated by
comparing the forward scatter (FSC) signals with those of
• Leads to minimal variation for consistent data acquisition the reference microspheres. The microspheres function as
reproducible size markers (Figure 7) and can be intermixed
• Compatible with any instrument with the experimental sample or used in parallel runs.

Flow cytometers are designed to perform quantitative Flow Cytometry Sub-micron Particle Size
measurements on individual cells and other particles with Reference Kit
high precision, speed, and accuracy. As with all high- The Invitrogen™ Flow Cytometry Sub-micron Particle Size
performance instrumentation, flow cytometers must be Reference Kit (Cat. No. F13839) provides a set of green-
calibrated frequently to ensure accuracy and reliability. fluorescent microsphere suspensions to serve as reliable
The stability, uniformity, and reproducibility of Invitrogen™ size references for flow cytometry users. The kit contains six
Molecular Probes™ microsphere products make them suspensions of polystyrene microspheres, each with a known
excellent tools for flow cytometer instrument set-up and diameter as determined by transmission electron microscopy.
calibration. Learn more about these Molecular Probes The excitation and emission profile of all the beads is similar to
products at thermofisher.com/flow-standards Invitrogen™ Alexa Fluor™ 488– or FITC-stained cells (excitation
and emission maxima are 505 nm and 515 nm, respectively).
Alignment
Number of particles
AlignFlow Cytometry Alignment Beads
Invitrogen™ AlignFlow™ Flow Cytometry Alignment Beads 102 103 104 102 103 104 102 103 104
are reliable references for aligning, focusing, and calibrating
flow cytometers. These fluorescently stained polystyrene Green Orange Red
microspheres are highly uniform with respect to size and
fluorescence intensity (Figure 6), and are designed to Fluorescence intensity
approximately replicate the size, emission wavelength,
and intensity of biological samples. Because the dyes are Figure 6. AlignFlow beads excited at 488 nm by an argon-ion laser
contained inside the microsphere’s matrix, instead of on the and monitored in three emission channels. Broad fluorescence emission
bead’s surface, AlignFlow beads have excellent photochemical is detected in all three channels. Note the exceptionally small variation in
and physical stability, providing reliable reference signals for
instrument set-up. The fluorescent dyes have been carefully fluorescence intensity of the beads. Data contributed by Carleton Stewart,
selected for optimal excitation by laser sources commonly
used in flow cytometry. The AlignFlow beads are available in Rosewell Park Cancer Institute.
three versions: for 350–370 nm excitation with UV lasers (Cat.
No. A16502, A16505), for 488 nm excitation with blue lasers 300 1.0 µm 6.0 µm 15 µm
(Cat. No. A16500, A16503), and for 633 nm excitation with red
lasers (Cat. No. A16501, A16504). Each version is available in Number of particles 2.0 µm 10 µm
two bead sizes: 2.5 μm diameter and 6.0 μm diameter.
200 4.0 µm

Size calibration 100

Flow Cytometry Size Calibration Kit 0
The Invitrogen™ Flow Cytometry Size Calibration Kit (Cat. 0 200 400 600 800 1000
No. F13838) provides a set of nonfluorescent microsphere
suspensions to serve as reliable size references for cytometry FSC
users. The kit contains six suspensions of unstained
polystyrene microspheres, each with a known diameter, Figure 7. Flow Cytometry Size Calibration Kit. Histogram analysis of the
forward scatter intensity (FSC) log channel values of the six polystyrene
microsphere samples supplied in the Flow Cytometry Size Calibration Kit
(Cat. No. F13838) is shown. FSC measurements were performed on a Becton
Dickinson FACScan™ flow cytometer using excitation at 488 nm.

6

The size (or size range) of bioparticles in an experimental during sorting). The beads can also be used to calibrate a
sample can be estimated by comparing their FSC with those flow cytometer’s laser source, optics, and stream flow without
of the reference microspheres (Figure 8). The microspheres in wasting valuable and sensitive experimental material.
each component function as reproducible size markers and
can be used individually (one size), premixed (two to six sizes), Compensation
intermixed with the experimental sample, or in parallel runs.
In a perfect world, the fluorescence emission profile for
This kit can be used to verify instrument performance and to each individual fluorophore would be a very intense, narrow
establish parameters that are suitable for analyzing sub-micron peak, well-separated from all other emission peaks. In
particles. For example, the kit can be used to check: reality, organic dyes and fluorescent proteins have broad
emission peaks. An example of the overlap of two commonly
• Resolution limit and dynamic range of particle size used fluorophores is shown in Figure 9, which features the
measurement emission profiles of Alexa Fluor 488 dye and R-phycoerythrin
(R-PE). For proper interpretation of the data collected, it is
• Sensitivity of forward and side scatter photomultiplier important to know that the fluorescent signal being
tubes recorded for Alexa Fluor 488 dye is, in fact, coming from
Alexa Fluor 488 dye and not from R-PE, which happens to
• Level of instrument baseline noise emit some light in the same wavelength range. To accurately
record the fluorescence signal for a given fluorophore, it is
• Laser and optical alignment and stability important to correct for the emission signal of all dyes, and
this correction is called compensation.
• Stability of the fluidics system

Cell sorting set-up

Cell Sorting Set-Up Beads We offer two types of compensation kits (Table 1). One
The Invitrogen™ Cell Sorting Set-Up Beads (Cat. No. C16506, type, the Invitrogen™ AbC™ bead kits, is designed for
C16507, C16508, C16509) are reliable standards for the set- compensation of dyes in immunophenotyping experiments
up and calibration of flow cytometry sorter instruments. The using fluorescently-labeled antibodies. The second type
beads have a diameter of 6 µm (±10%), and thus approximate of compensation kit, the Invitrogen™ ArC™ Amine Reactive
the size, emission wavelength, and intensity of many biological Compensation Bead Kit, is designed for use with cell viability
samples. Consequently, the beads can be used to check cell assays that use amine reactive dyes, such as the Invitrogen™
sorter settings such as drop delay and efficiency (cell loss LIVE/DEAD™ Fixable Dead Cell Stain Kits (see “Cell viability” on
page 20 for more details on these assays).

106 2 µm 100
105 1 µm

Side scatter

Relative intensity (%)
104 75
0.5 µm 50
25
103 0.2 µm

0.1 µm
102

101

100 103 104 105 106 0
102 300
Green uorescence
400 500 600 700 800 900
Wavelength (nm)

Figure 8. Flow Cytometry Sub-micron Particle Size Reference Kit. Signals Figure 9. Emission profiles of Alexa Fluor 488 dye (green curve) and R-PE
from (the kit has six particle sizes; only five were used here) five different- (orange curve).
sized particles of the Flow Cytometry Sub-micron Particle Size Reference Kit
(Cat. No. F13839) were acquired using 488 nm excitation and a 530/30 nm
bandpass (BP) emission filter on the Attune flow cytometer. The diameters
of the five different green-fluorescent microspheres are identified on a plot of
particle fluorescence versus side scatter.

7

Table 1. Compensation kit selection guide.

Product name Assay type Positive bead Negative bead Cat. No.
No binding capacity
AbC Total Antibody Compensation Immunophenotyping Hamster, mouse, rabbit, and rat antibodies* A10497,
Bead Kit Mouse monoclonal antibodies* No binding capacity A10513
No binding capacity A10344
AbC Anti-Mouse Bead Kit Immunophenotyping No amine reactive
capacity A10389
AbC Anti-Rat/Hamster Bead Kit Immunophenotyping Rat and hamster monoclonal antibodies* A10346
Cell viability assay
ArC Amine Reactive Amine reactive dyes and LIVE/DEAD Fixable
Compensation Bead Kit Dead Cell Stains

Set-up and calibration *AbC capture beads bind all isotypes.
All products in this table are RUO, For Research Use Only. Not for use in diagnostic procedures.

AbC compensation bead kits ArC Amine Reactive Compensation Bead Kit
• Eliminates the hassle of heat-treating cells
• An alternative to using precious samples for setting flow
cytometry compensation • Optimized for all LIVE/DEAD Fixable Dead Cell Stain kits

• Highest reactivity to different subclasses of mouse, rat, • Fast and simple bead-based flow cytometry
and hamster immunoglobulin compensation

• Fast and simple bead-based flow cytometry • An alternative to using precious samples for setting
compensation compensation

• Removal of inconsistencies due to variations in antigen • Enables accurate and consistent results
expression

AbC bead kits provide a consistent, accurate, and simple-to- The ArC Amine Reactive Compensation Bead Kit (Cat. No.
use technique for the setting of flow cytometry compensation A10346) provides a consistent, accurate, and simple-to-use
when using 1) fluorophore-conjugated hamster, mouse, rabbit, technique for the setting of flow cytometry compensation
or rat antibodies (AbC Total Antibody Compensation Bead Kit, when using any of the LIVE/DEAD Fixable Dead Cell Stain kits
Cat. No. A10497, A10513; Figure 10); 2) using fluorophore- or when using any amine reactive dye. LIVE/DEAD Fixable
conjugated mouse antibodies (AbC Anti-Mouse Bead Kit, Cat. Dead Cell Stain kits (and amine reactive dyes) can be used to
No. A10344, Figure 11); or 3) using fluorophore-conjugated evaluate mammalian cell viability based on the fact that the
rat or hamster antibodies (AbC Anti-Rat/Hamster Bead Kit, dye reacts with cellular amines. The reactive dye can enter
Cat. No. A10389). All three kits contain two types of specially the cell via compromised membranes of necrotic cells and
modified polystyrene microspheres: 1) AbC capture beads react with free amines in the interior and on the surface of the
(also called positive beads), which bind all isotypes of the cell, resulting in intense fluorescent staining. In contrast, only
specific immunoglobulin, and 2) negative beads, which the cell-surface amines of viable cells are available to react
have no antibody binding capacity. After incubation with a with the dye, resulting in relatively dim staining. The difference
fluorophore-conjugated primary antibody (hamster, mouse, in fluorescence intensity between the live and dead cell
rat, or rabbit, depending on the kit used), the two components populations is typically greater than 50-fold.
provide distinct positive and negative populations of beads
that can be used to set compensation (Figure 11). The ArC Amine Reactive Compensation Bead Kit includes
two types of specially modified polystyrene microspheres to
allow easy compensation of the LIVE/DEAD Fixable Dead Cell
Stains: the ArC reactive beads (Component A), which bind
any of the amine reactive dyes, and the ArC negative beads
(Component B), which have no reactivity. After incubation
with any amine reactive dye, the two kit components provide

8

distinct positive and negative populations of beads that can m90o0 useMoausenIgtGi1bodies, allowing even m50o0 re cRaot IgnG1sistent and
be used to set compensation (Figure 12). The ArC Amine 800 Mouse IgG2a Rat IgG2a
Reactive Compensation Bead Kit can be combined with the
AbC Anti-Mouse Bead Kit for use with fluorophore conjugated Number of eventsaccurate700 Mouse IIcggGGo32bmpensation for multico4l0o0 r Number of events imRRaamtt IIggGGu22cbnophenotyping
600 Mouse
ex50p0 erimMouesenIgGtMs that also incorporate a300LIVERat/IgDGMEAD Fixable Dead
Cell400
300 Stain. 200

200 100
100

0 0 102 103 104 105 106
101 102 103 104 105 106 101

A B C PE uorescence D PE uorescence

900 Mouse IgG1 500 Rat IgG1 600 700 Rabbit monoclonal IgG
800 Mouse IgG2a Rat IgG2a Armenian hamster IgG Rabbit polyclonal IgG
700 Mouse IgG2b 400 Rat IgG2b
600 Mouse IgG3 Rat IgG2c 500 Syrian hamster IgG 600
500 Mouse IgGM 300 Rat IgGM
Number of events 400 Number of events Number of events 400 500 Number of events
300 102 103 104
200 200 300 400
100 PE uorescence
100 300
0 200
101
200

100 100

105 0 106 0 0
106 101 102 103 104 105 101 102 103 104 105 106 101 102 103 104 105 106

PE uorescence PE uorescence PE uorescence

600 Armenian hamster IgG Rabbit monoclonal IgG

Figu50r0e 10S.yrHianihsatmostegr IrgaG ms showing the staining700of thReabbAit pbolyCclonTaol IgtGal Antibody Compensation Bead Kit. Signal separation of the positive capture beads for mouse
(A), rat (B), and hamster (C) monoclonal antibo60d0ies, and rabbit (D) monoclonal and polyclonal antibodies. Beads were labeled with an optimized amount of each PE
Number of eventsanti4b00ody conjugate and analyzed on an Attune500cytometer using 488 nm excitation and a 574/26 nm bandpass filter.Number of events
300 400

300
200

200

100 100 B 250 C
0 105
A 250 101 102 103 104 105 106
0
101 102 103 104 105 106

200 PE uorescence PE uore2sc0e0nce CD3-FITC uorescence

Events counted 150 150 Events counted 104

100 100 103

50 50 102
0
0 -102
-350 -102 0 102
-102 0 102 103 104 105 103 104 105 -102 0 102 103 104 105
PE uorescence
FITC uorescence CD56-PE uorescence

Figure 11. Compensation using the AbC Anti-Mouse Bead Kit. (A) Phycoerythrin (PE)-conjugated mouse anti-human CD56 antibody (Cat. No. MHCD5604) was
used with AbC capture beads for a positive signal and with negative beads for a negative signal. (B) FITC-conjugated mouse anti-human CD3 antibody (Cat. No.
MHCD03014) was used with AbC capture beads for a positive signal and with negative beads for a negative signal. (C) Dual-parameter plot showing gated human
lymphocytes labeled with PE–conjugated mouse anti-human CD56 and FITC-conjugated mouse anti-human CD3 antibodies after compensation was performed

with the AbC Anti-Mouse Bead Kit (Cat. No. A10344).

A 400 Events counted B C 350
350 Events counted 350 300
300 Events counted300 250
250 250 200
200 200 150
150 150 100
100 100

50 50 50

0 103 104 105 0 103 104 105 0 103 104 105
-102 0 102 -288 -102 0 102 -715 -102 0 102

LIVE/DEAD™ Fixable Violet stain uorescence LIVE/DEAD™ Fixable Green stain uorescence LIVE/DEAD™ Fixable Red stain uorescence

Figure 12. Staining profile of the ArC Amine Reactive Compensation Bead Kit components with 3 LIVE/DEAD Fixable Dead Cell Stain kits. (A) LIVE/DEAD Fixable
Violet dye stained beads (Cat. No. L34955) were analyzed with 405 nm excitation, emission was collected with a 450/50 nm bandpass filter. (B) LIVE/DEAD Fixable Green
dye stained beads (Cat. No. L23101) were analyzed with 488 nm excitation, emission was collected with a 525/50 nm bandpass filter. (C) LIVE/DEAD Fixable Far Red dye
stained beads (Cat. No. L10120) were analyzed using 633 nm excitation, emission was collected with a 660/20 nm bandpass filter.

9

Table 2. Absolute counting beads selection guide.

Product name Sample Bead size Excitation Emission Parameters Regulatory Cat. No.
type 7 μm (nm) max (nm) measured status* C36950
CountBright Absolute RUO PCB100
Counting Beads Any type UV to 635 385 to 800 Number of cells
RUO
AccuCheck Counting Whole Bead A: 6.40 μm Bead A: 488 Bead A: 575–585 Number of cells and
Beads blood Bead B: 6.36 μm Bead B: 635 Bead B: 660–680 accuracy of pipetting

*For Research Use Only. Not for use in diagnostic procedures, unless otherwise indicated.

Set-up and calibration Absolute cell counting For absolute counts, a specific volume of the microsphere
suspension is added to a specific volume of sample, so that
Flow cytometry provides a rapid method to quantify cell the ratio of sample volume to microsphere volume is known.
characteristics. However, most flow cytometers cannot directly The volume of sample analyzed can be calculated from the
provide the cell concentration or absolute count of cells in number of microsphere events, and can be used with cell
a sample. Absolute cell counts have been widely used in events to determine cell concentration (Figure 13). In general,
quantifying cell populations and disease progression, including at least 1,000 bead events should be acquired to assure a
in studies of stem cells. Absolute cell counts are generally statistically significant determination of sample volume.
obtained either by combining a separate cell concentration
determination from a hematology analyzer with flow cytometric CountBright Absolute Counting Beads can be used with
population data (multiple platform testing) or by adding an any sample type, including lysed/no-wash whole blood.
internal microsphere counting standard to the flow cytometric The microspheres in the reagents are approximately 7 µm in
sample (single platform testing). The single platform method diameter and have settling properties similar to lymphocytes.
is preferred as it is technically less complicated and it avoids Sample preparation steps that can lead to cell or microsphere
interlaboratory variation and underestimations, making it more loss, such as washes, should be avoided. CountBright beads
accurate than multiple platform testing. can be used with either a scatter or fluorescence threshold.
When using a scatter threshold, the microsphere signal should
We offer two products for cell counting—Invitrogen™ be above the threshold. The microspheres can be gated by
CountBright™ Absolute Counting Beads and Invitrogen™ a single parameter, but a combination of parameters can be
AccuCheck™ Counting Beads. See Table 2 to determine used to resolve microspheres from cells and other events.
which kit is right for your experiment.

CountBright Absolute Counting Beads AccuCheck Counting Beads

• Compatible with every commercially available flow • Internal control using ratio of two different color beads
cytometer because they are loaded with a wide breadth indicates pipetting accuracy
of fluorochromes
• Single platform is preferred over multiple platform testing,
• Easy-to-use protocol that works with multiple cell types to enable consistency in results
including lysed/no-wash whole blood
• Easy to validate with most immunophenotyping
• More reliable than multiple platform testing experiments

CountBright Absolute Counting Beads (Cat. No. C36950) AccuCheck Counting Beads (Cat. No. PCB100) are an
are a calibrated suspension of microspheres that are brightly efficient single platform method for absolute cell counting
fluorescent across a wide range of excitation and emission that combines the advantages of direct flow cytometric
wavelengths (UV to 635 nm excitation and 385 to 800 nm immunophenotyping with the use of two different fluorescent
emission) and contain a known concentration of microspheres. beads (A and B beads). These two fluorospheres are used
as a double internal standard for blood volume calculation.

10

A known volume of AccuCheck Counting Beads is added to Figure 13. CountBright Absolute Counting Beads. A mixture of live and
the same known volume of stained blood in a lysed/no-wash heat-killed Jurkat cells were treated with reagents in the LIVE/DEAD Viability/
technique. The beads are counted along with cells. Because Cytotoxicity Kit (Cat. No. L3224). CountBright Absolute Counting Beads (Cat.
the concentration of beads is known, the number of cells No. C36950) were added to the sample, which was then analyzed by flow
per microliter (the absolute count) is obtained by relating the cytometry using 488 nm excitation. Calcein fluorescence was collected with a
number of cells counted to the total number of fluorescent 530/30 nm bandpass filter and ethidium homodimer-1 (EthD-1) fluorescence
bead events. The cell number is then multiplied by the number was collected with a 610 nm longpass filter. The data show clear separation of
of total fluorospheres per unit of volume. As the AccuCheck live and dead cells, as well as separation of the counting beads.
Counting Beads system contains two different fluorospheres
in a known proportion, the accuracy of the assay pipetting can
be verified using the proportion of both types of beads.

Instrument set-up and calibration product list.

Section Product name Laser type Ex/Em* Regulatory Size Cat. No.
status†
Alignment AlignFlow Flow Cytometry Alignment Beads UV 350–370/ 3 mL A16502
for UV Lasers, 2.5 μm 400–470 RUO
Size calibration Blue 3 mL A16505
Cell sorting AlignFlow Flow Cytometry Alignment Beads 488/515–660 RUO
set-up for UV Lasers, 6.0 μm Red 3 mL A16500
Compensation 633/645–680 RUO
AlignFlow Flow Cytometry Alignment Beads NA 1 kit A16503
for Blue Lasers, 2.5 μm Blue NA RUO 1 kit
UV 505/515 RUO 3 mL A16501
AlignFlow Flow Cytometry Alignment Beads Blue 350–375/460 RUO 3 mL
for Blue Lasers, 6.0 μm Green-yellow 488/515 RUO 3 mL A16504
Red 532, 561/575 RUO 3 mL
AlignFlow Flow Cytometry Alignment Beads NA RUO 100 tests F13838
for Red Lasers, 2.5 μm NA 633/680 RUO 25 tests
NA NA RUO 1 kit F13839
AlignFlow Flow Cytometry Alignment Beads NA NA RUO 1 kit
for Red Lasers, 6.0 μm Blue/red NA RUO 1 kit C16506
UV to red NA RUO 10 mL C16508
Flow Cytometry Size Calibration Kit RUO 5 mL C16509
(nonfluorescent microspheres) 488/575-585 (Bead A)
635/660–680 (Bead B) C16507
Flow Cytometry Sub-micron Particle Size UV to 635/385 to 800 A10497
Reference Kit A10513
A10344
Cell Sorting Set-Up Beads for UV Lasers A10389
A10346
Cell Sorting Set-Up Beads for Blue Lasers PCB100

Cell Sorting Set-Up Beads for Green-Yellow C36950
Lasers

Cell Sorting Set-Up Beads for Red Lasers

AbC Total Antibody Compensation Bead Kit

AbC Anti-Mouse Bead Kit

AbC Anti-Rat/Hamster Bead Kit

ArC Amine Reactive Compensation Bead Kit

Absolute cell AccuCheck Counting Beads
counting CountBright Absolute Counting Beads

NA, not applicable.
*Excitation and emission maximum wavelengths, in nm.
†RUO, For Research Use Only. Not for use in diagnostic procedures.

11

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Side scatter B220 Pacific OrangeTM
SidSeidsceastctaetrter B22B022P0acPifaiccifiOcraOnrgaenTMgeTM
SidSeidsceastctaetrter B22B022P0acPifaiccifiOcraOnrgaenTMgeTM
puchtrioleitlmiyfeforoakrtiniooennsc,,oaalnondgd••ys,gCDbatneieroimsond••nmtcwivpmnaDbCheocnetctnoiwusothndniietepvnecivplnenfhrlesoaoctftnarpuwloctseociailepviteqszcgfeonroeuseetyfrra—piuas,sscauliicr,tcqzcsfcioooeueaeemhrn—irslsl.suipactsswFilnoooeiefdgmrnteroaensalpalmllnsalnoaeeafdltixlsnaseyupalsugpelansisrbee,anfiitxlestsatyupsnoiseppbefciosttroofenwiieistpddlsaenoetirvefvceooeteennws—nsltslaeieiwsisfnrvvim,eecteiut—nlaahsitlstilinivmoteunaitllivfrcbleuyeuntoodnrmecbrfoinltuyepuegnthcotodrn(tormeTyoirnatdepteebgoshctwlr(metoaTiyetrna1dhaet)bdot.swaclMfmenhalitunu1haoto)hdltp.tarceiMtfepnhilopsulueotncaohlpeatnfreiltnlepeuiop5tslloesnc6apr1eanfryelnoloesunt5ulcomes6peria1nnryerloostasnetucspmueceeriasunnorerlrstae(derFspueiciinernngsuorseutrftl(drcleryFoeeaiiinwnngn2sut)fl.rclyoeawn2). Counts C CC
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Antigen detection
of heterologous cell populations to rare-event detection, ForwardFsocrawtateFrrdorswcatrtderscatter CD45.2 ACPDC4-5C.2Cy®AD7P45C.-2CAyP®7C-Cy®7 CD3 PerCCDP3-CPyeC®r5DC.35PP-CeyrC®5P.-5Cy®5.5
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multicolor flow cyAtomA etry with Molecular Probes fluorescentlyB BB B C CC
BB CC
B220 Pacific OrangeTM CD8 Alexa Fluor® 700 BB MHCII Pacific BlueTM
conjugated primary antibodies can help answer complex cell B220 Pacific OrangeTM CD8CDAl8eAxlCaeoxFulanutFolsru®o7r®00700 MHMCIIHCPIIacPifaiccifiBlcuBelTMueTM CD8 Alexa Fluor® 700
Side scatter CCDD8C8DAAll8eAxlCaeoxFulanutFoolsrru®7o07r0®00700 CD25 APC MHMCIIHCPIIacPifaiccifiBlcuBelTMueTM ®
Side scatter CD2C5D2A5PACPC
biology questions—in less time and with less sample than CC CD2C5D2A5PACPC
other approaches. Search for your primary antibody conjugate

at thermofisher.com/flow-searchantibodies CD45.2CADP4C5-.C2yA®P7 C-Cy®7 CD4 PacCiCfDiDc43GPPraeCcerCDiCnfD4iTPcM3P-GPCarecyeir®feC5icn.P5TGM-rCeye®n5T.M5 Foxp3 PEFoxp3 FPoExp3 PE CD11c PEC-DC1y1®c7CPDE1-1Ccy®P7E-Cy®7
CD4 PacCiDfic4 GPraeceifnicTMGreenTM Foxp3 PFEoxp3 PE CD11c PCED-1C1yc®7PE-Cy®7
ForwarFdosrcwaattredrscatter

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technology. We are able to offer a completeCD4 PacCiDfic4GPraeceifnicTMGreenTM portfoFolixop3 ofPFEoxp3 PE CD11c PCED-1C1yc®7PE-Cy®7 CD11b FCITDC11b FITC

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visible, and near-IR spectrum. This expertise extends to ourebWxaipsthreeidnsebWostxahniptstehhereBixdnsp2tosrtr2hnaet0enshe–ss,eBxiCcpo2trDrrn2iaep40onst5–ifss,o.ti2Ccnoh+rDneifgpa4aoctc5toifote.-to2n,hrr+TefgFaccacoecetoxtpel-oplt,rsro3TerFwcacsoeneCxprdlpleDtsto3h4arwaensonearcCrdleCyeDltzDlh4aese8ndouar(cbrBleCyfa,alzDsllceees8edfdut(mbr)Bo.fan,aWslrcteeikehtfdehtem)rioi.nrnCaWetrDthxikht2epehe5rrCiiner(CDIsetLD4sh-xi+2epoTR5rnCeαc(DoIsCs)eLf4s(lu-B5lCi+2sorT,RD7nftraα3chBiog)ec(fLh(lAreBlCets/,),D6.riatrsiC3higngsaeDh(htArpt3siet),gu–).l.ericbCesiCegnpDnaDlhool3sts3sp+)cu–.wTlcybCilseacptDtrteleoilee3olspsl+ndssuwTewloweapicfnetreasierolrurTleansespatespoewepbsfrdepaeuslrtearsuorearspfats4siapevhte.reecdopedTerwtasoeihrsnagsasiitvuhsttoreealaoatdsrrwiwtneeotinoeagerptuypporodloaTpatprwwuwtceuoleoaarlipltatyplsohitsooTpiontpfupchonuleasrallllitatlomitsoiwontahnerasydt
antibodies to the
by fixation and
primary and secondary antibody conjugation capabilities,of CD1o1fc+CMD1H1CcI+IM+, pHrCoIfIe+,spsrionfeaslsainontiagleann-ptirgeesne-nptriensgednetnindgridtiecncderliltsic(Cce, llesft()C. ,Slpelfet)n.iScppdelernmdicreidtaiecnbcdielrilizltsiacctcaioenlnlbseocsafuntbhbdeeivsisdueabdmifvupidrlteehdeufrusirnitnthogeCrDtinh1t1eobC+FaDInX1d1b&C+DaP8nE+d RCDM8+Cell Permeabilization Kit
resulting in brightdeenrd,rmidtiecnocderrlieltiscuscbetslael tsbsu(blCes,ertfislguh(Cto),,rreiegahcsht)c,peoeascnshetspsocisnosgenusnsjuinquggeuaantniqetuigsee.annptrigeesnenptraetsioenntparto(ioCpnearpttr.ieoNsp.eor.tieGs.AS003, GAS004). Intracellular staining was performed during the
permeabilization step using PE-conjugated rat anti-mouse Foxp3 antibody.
Regardless of your instrument, we have labels designed to Lymphocytes were gated using FSC/SSC parameters (A, left) and B220-

help you get the most out of every sample and every flow expressing B cells were omitted from subsequent analysis (A, middle). Within
cytometry run. See Table 3 for a list of available fluorophores the B220-, CD45.2+ gate, T cells were analyzed based on their expression

of CD3 (A, right). CD3+ T cells were separated into two populations based

and their specifications. on expression of the co-receptors CD4 or CD8 (B, left). Within the CD4+ T
cells there is a subpopulation of suppressive regulatory T cells that express

the transcription factor Foxp3 and the cell surface marker CD25 (IL-2Rα)
(B, right). CD3- cells were separated to show a rare population of CD11c+

MHCII+, professional antigen-presenting dendritic cells (C, left). Splenic

dendritic cells can be subdivided further into CD11b+ and CD8+ dendritic cell

subsets (C, right), each possessing unique antigen presentation properties.

12

Table 3. Fluorophores for flow cytometry.

Fluorophore Laser Emission (nm)

Antibody conjugates UV Violet Blue Green Yellow Red
Alexa Fluor 350 405 nm 488 nm 532 nm 561 nm 633/635 nm
Alexa Fluor 405
Pacific Blue 442
Pacific Green 421
Pacific Orange 455
Alexa Fluor 488 500
Fluorescein (FITC) 551
Qdot 605 519
Qdot 655 525
Qdot 705 605
Qdot 800 655
Peridinin chlorophyll (PerCP) 705
PerCP-Cy5.5 800
R-phycoerythrin (R-PE, PE) 678
PE-Texas Red 695
PE-Alexa Fluor 610 575
TRI-COLOR (TC, PE-Cy5) 615
PE-Cy5.5 628
PE-Alexa Fluor 700 670
PE-Cy7 694
Allophycocyanin (APC) 723
APC-Cy5.5 767
APC-Cy7 660
APC-Alexa Fluor 750 694
Alexa Fluor 647 767
Alexa Fluor 700 775
APC-Alexa Fluor 750 668
719
775

Table 4. Products used in multiparametric analysis of T cells and dendritic cells (Figure 14).

Target* Host Fluorophore Laser Emission max (nm) Cat. No.
Violet 455 A14901
MHCII Rat Pacific Blue dye Violet 500 C11207
Violet 551 RM2630
CD4 Rat Pacific Green dye Blue 525 RM2801
Blue 575 N/A
CD45R Rat Pacific Orange dye Blue 695 A14784
Blue 767 A15849
CD11b Rat FITC Red 660 NA
Red 767 A18642
Foxp3 Rat R-PE Red 719 MCD0829

CD3 Hamster PerCP-Cy5.5

CD11c Hamster PE-Cy7

CD25 (IL-2Rα) Rat APC

CD45.2 Mouse APC-Cy7

CD8 Rat Alexa Fluor 700

*All products are For Research Use Only. Not for use in diagnostic procedures.
NA-not available for purchase

13

Qdot antibody conjugates conjugates, Qdot antibody conjugates can be excited with
• Excited by 405 nm or 488 nm, maximizing violet laser use any wavelength below their emission maximum, but are best
excited by UV or violet light. The narrow, symmetric emission
• Combine with existing organic dyes, increasing the profiles of Qdot antibody conjugates allow for minimal
number of detectable parameters compensation when using a single excitation source, and their
very long Stokes shifts enable better, more efficient multicolor
• Do not degrade over time like tandem conjugates, assays using the 405 nm violet laser. Available in multiple
allowing greater reproducibility colors for use in flow cytometry (Figure 15 and Table 5), these
advantages make Qdot antibody conjugates powerful tools for
• Narrow emission spectra allow for minimal compensation antibody labeling and staining.
when using a single excitation source
Learn more about Qdot nanocrystals and their applications in
Qdot antibody conjugates possess a bright fluorescence flow cytometry at thermofisher.com/flow-qdot
emission that makes them well suited for the detection of
low-abundance extracellular proteins. Approximately the same
size as R-PE and compatible with existing organic fluorophore

Antigen detection Table 5. Qdot primary antibody conjugates selection guide.

Target* Clone Qdot 605 Qdot 655 Conjugate‡ Qdot 800
Qdot 705 Q22142§
Q22149§
CD2 S5.5 Q10172 Q22140§ Q22141§
Q22153§
CD3e S4.1 (AKA 7D6) Q10012
Q10484§ Q22165§
Q22157§
CD3e S3.5 Q10054
Q10064
CD4 RM4-5 Q10008 Q10007 _Q10060 Q22139§
Q10480§ Q10482§ Q10485§ Q22162§

CD4† 3B5 Q10092 Q22163§ Q22164§ Q22152§
Q10156
CD8 MEM-78 Q10009 Q10055 Q10059 Q22168§
Q10481§ Q10483§ Q22156§
Q10063
CD10 TüK4 Q10481§" Q22136§

CD14 SJ25-C1 Q10053

CD14 6D5 Q10013 Q10056 Q22137§

CD19 CLB-27/1 Q10306 Q10179 Q22138§

CD19† HIT2 Q22160§ Q10379 Q22161§

CD27 H130 Q10065 Q10066

CD38 RA3-6B2 Q22150§

CD45 MEM-56 Q10051 Q22154§ Q10062

CD45R† Tü36 Q22166§ Q10176 Q22167§

CD45RA Q10047 Q10069 Q22155§

HLADR (class II) Q10052 Q22158§ Q22159§

Isotype control mouse IgG2a Q10014 Q10015 Q10076

*Host for all antibodies is mouse unless otherwise indicated. All products are For Research Use Only. Not for use in diagnostic procedures.
†Host is rat.
‡All product sizes are 100 tests unless otherwise indicated.
§Product size is 25 tests.

14

A 7 6 58 B 700

5,000,000 1. Qdot® 525 1 2 34 5 6 7 8
2. Qdot® 565
Extinction coef cient (cm–1M–1)4,000,0003. Qdot® 585 600
Fluorescence emission4. Qdot® 605
3,000,000 4 5. Qdot® 625 500
2,000,000 3 Number of cells6. Qdot® 655
7. Qdot® 705
8. Qdot® 800

1,000,000 2 400
1 300

0 200
400 450 500 550 600 650 700 750 800
450 500 550 600 650 700 750 800 850 900 100
Wavelength (nm)
Wavelength (nm) 0
102
Figure 15. Absorption and emission profiles of Qdot labels. 103 104 105 106
(A) Absorption spectra of Qdot labels plotted in terms of molar extinction
coefficient. (B) Normalized emission spectra of Qdot labels. Numbers are the Paci c Blue uorescence
same as those in panel A.
Figure 16. Histogram of gated U2OS cells expressing mCherry protein and
mCherry rat monoclonal antibody conjugates
Fluorescent conjugates of our affinity purified rat monoclonal labeled with Pacific Blue dye-conjugated rat anti-mCherry antibody (Cat.
antibodies for mCherry fluorescent protein (Table 6) can No. M11238). Samples were acquired and analyzed using 405 nm excitation
be used to detect native and denatured forms of mCherry and 522/31 nm band pass emission filters on an Attune flow cytometer.
or mCherry fusion proteins in flow cytometry applications
(Figure 16). Full-length mCherry was used as the Table 6. mCherry rat monoclonal antibody
immunogen, and the resulting monoclonal IgG2a-isotype (clone 16D7) conjugates.
antibody detects denatured and native forms of the protein.
Because of its improved brightness, superior photostability, Dye conjugate Excitation Excitation/ Cat. No.
and extremely rapid maturation rate, mCherry monomeric red Pacific Blue laser color Emission (nm)
fluorescent protein is becoming the red fluorescent protein of M11238
choice for monitoring physiological processes and detecting Violet 405/455 M11239
transgene expression. M11240
Alexa Fluor 488 Blue 488/519 M11241

Alexa Fluor 594 Red 590/617

Alexa Fluor 647 Red 650/668

Antibody labeling

We offer a number of Molecular Probes labeling kits for the
direct attachment of intensely fluorescent Alexa Fluor dyes,
Qdot nanocrystals, R-phycoerythrin (R-PE), or even biotin to
IgG antibody at levels less than 10 μg up to 1 mg. Directly
labeled antibodies allow you to use more than one same-
species antibody in a single staining experiment. You can use
traditional labeling chemistries optimized for your application
or site-specific labeling using click-chemistry technology.
Below we describe two options for easy labeling of flow
cytometry primary antibodies. To find more information and
products, go to thermofisher.com/flow-antibodylabeling

15

The SiteClick antibody labeling system Antibody
• Contains reagents to label 100 µg of IgG antibody • Polyclonal
• Monoclonal
• Easy-to-follow step-by-step protocol • Recombinant

• Highly efficient site-specific, reproducible labeling
chemistry

• R-PE and Qdot labels for flow cytometry

Antigen detection The Invitrogen™ SiteClick™ system represents a new paradigm β-Galactosidase
for the universal site-selective labeling of antibodies. This GalT(Y289L)
modular, click chemistry–mediated method allows you to UDP-GalNAz
enzymatically label essentially any antibody on its heavy chain
N-linked glycans. In contrast to standard antibody labeling N3 N3
techniques, which can be tedious and inconsistent, the N3 N3
SiteClick site-selective approach produces highly robust and
reproducible labeling of antibodies with an impressive choice Azide-activated antibody
of detection molecules.
DIBO alkyne label
The site-selective labeling provided by the SiteClick system
(Figure 17) prevents disruption of the antigen-binding domain Site-selectively
that can occur with traditional amine or thiol reactive labeling labeled antibody
reagents and eliminates the need to genetically engineer Figure 17. The SiteClick antibody labeling system. The first step in the
labeling sites into the antibody prior to modification. This SiteClick antibody labeling process involves removal of terminal galactose
site-selective strategy is especially important when labeling residues from heavy chain N-linked glycans using β-galactosidase, exposing
monoclonal antibodies that contain lysine residues in or essentially all possible modifiable GlcNAc residues. Second, the free terminal
around the antigen-binding domain, as labeling of these sites GlcNAc residues are activated with azide tags by enzymatic attachment of
can disrupt antigen binding. Monoclonal antibodies can also GalNAz to the terminal GlcNAc residues using the GalT(Y289L) enzyme. In the
have variable sensitivity to disulfide bond–reducing agents third step, the azide residues are reacted with the dibenzocyclooctyne (DIBO)-
used in reductive cysteine labeling, leaving partially dissociated functionalized probe of choice (e.g., Alexa Fluor 488 DIBO alkyne). The average
antibody fragments that result in decreased antibody binding degree of labeling is 3–3.5 labels per antibody.
and yield.

We offer antibody labeling kits with R-PE and an array of
Qdot labels (Table 7), which are designed especially for flow
cytometry applications. The kits are configured to provide
an easy-to-follow workflow designed to allow novice and
experienced scientists alike to obtain efficient antibody
labeling every time (Figure 18). And, because antibody glycans
are highly conserved, even between different species, the
reproducibility of SiteClick labeling for different antibodies
is very high, precluding the need to optimize labeling of
each newly acquired antibody. To find out more, go to
thermofisher.com/flow-siteclick

16

Zenon labeling technology CD4-positive cells (%) 60
Invitrogen™ Zenon™ labeling technology provides a versatile, easy-to-use system Novice
for labeling mouse IgG1, IgG2a, and IgG2b isotypes, as well as rabbit, goat, and Expert
human IgG antibodies, with our premier Molecular Probes dyes as well as other Reference R-PE
fluorophores, biotin, photoproteins, and enzymes. This technology offers several
advantages over direct chemical labeling, including: 40

• Speed—the entire labeling procedure typically takes only 10 minutes 20

• Efficiency—designed to label nearly 100% of the primary antibodies in solution 0 SiteClick™
SiteClick™ R-PE
• Economy—submicrogram amounts of antibody can be labeled Qdot™ 605

• Simplicity—no pre- or postlabeling purification of the antibody is required Figure 18. Efficient antibody labeling for both
novice and experienced scientists. The SiteClick
• Flexibility—easily use multiple primary antibodies in a single experiment Qdot 605 Antibody Labeling Kit and SiteClick
R-PE Antibody Labeling Kit were used by both
Zenon labeling technology uses a fluorophore-, biotin-, or enzyme-labeled Fab a novice user and an expert user to label an
fragment directed against the Fc portion of an intact IgG antibody to form a anti-CD4 monoclonal antibody in triplicate. The
noncovalent labeling complex. To help ensure their high affinity and selectivity for labeled antibodies were used to label CD4-positive
the Fc portion of the target antibody, the Zenon labeling reagents have been affinity cells isolated from a single human blood donor,
purified during their preparation. Because the Zenon labeling method is based with subsequent analysis by flow cytometry; the
on immunoselectivity, there is no need to remove exogenous proteins or amine reference sample is a commercially available R-PE
containing buffers from the antibody sample prior to forming the complex. Antibodies anti-CD4 antibody conjugate. The data show
labeled using Zenon technology display fluorescence intensity or enzymatic activity percentages of CD4-positive cells relative to total
similar to that observed for directly labeled antibody conjugates. cells, and the error bars indicate variation in the
triplicate kit labelings for each user. The novice user
had never before performed protein bioconjugation,
yet obtained labeling efficiencies equivalent to
those obtained by an expert user.

Our wide selection of Zenon labeling reagents (Table 8) can be mixed and matched,
providing the freedom to experiment with multiple dye–antibody combinations in flow
cytometry applications. Learn more about the Zenon antibody labeling technology
and other antibody labeling products at thermofisher.com/flow-antibodylabeling

Table 7. SiteClick antibody labeling kits. Excitation laser color Emission (nm) Cat. No.
Blue 578 S10467
Product UV, violet, blue 525 S10449
SiteClick R-PE Antibody Labeling Kit UV, violet, blue, green 565 S10450
SiteClick Qdot 525 Antibody Labeling Kit UV, violet, blue, green, yellow 585 S10451
SiteClick Qdot 565 Antibody Labeling Kit UV, violet, blue, green, yellow 605 S10469
SiteClick Qdot 585 Antibody Labeling Kit UV, violet, blue, green, yellow 625 S10452
SiteClick Qdot 605 Antibody Labeling Kit UV, violet, blue, green, yellow 655 S10453
SiteClick Qdot 625 Antibody Labeling Kit UV, violet, blue, green, yellow, red 705 S10454
SiteClick Qdot 655 Antibody Labeling Kit UV, violet, blue, green, yellow, red 800 S10455
SiteClick Qdot 705 Antibody Labeling Kit
SiteClick Qdot 800 Antibody Labeling Kit

17

Table 8. Zenon labeling kit selection guide.

Dye Ex/Em* Mouse IgG1 Mouse IgG2a Mouse IgG2b Rabbit IgG Goat IgG Human IgG
Alexa Fluor Dyes† Z25402
Alexa Fluor 350 346/442 Z25000 Z25100 Z25213 Z25300 Z25602 Z25407
Alexa Fluor 405 402/421 Z25013 Z25113 Z25202 Z25313 Z25605 Z25408
Alexa Fluor 488 495/519 Z25002 Z25102 Z25302 Z25607
Alexa Fluor 546 556/573 Z25004 Z25205 Z25304 Z25608 Z25452
Alexa Fluor 555 555/565 Z25005 Z25105 Z25305 Z25455
Alexa Fluor 568 578/603 Z25006 Z25106 Z25207 Z25306 Z25451
Alexa Fluor 594 590/617 Z25007 Z25107 Z25208 Z25307
Alexa Fluor 647 650/668 Z25008 Z25108 Z25210 Z25308
Alexa Fluor 680 679/702 Z25010 Z25110
Alexa Fluor 700 696/719 Z25011
Classic Dyes†

Pacific Blue 410/455 Z25041 Z25156
Z11203 Z25257
Pacific Green 411/500 Z25256
Z25042 Z25152
Antigen detection Pacific Orange 400/551 Z25155
Z25052
Fluorescein 494/518 Z25151 Z25342
Z25055 Z25352
Biotin† Z25020 Z25355
Z25021
Biotin-XX NA Z25051 Z25252 Z25351
Z25031 Z25255
Phycobiliproteins and Tandem Dyes‡
Z25251
R-PE 496§/578

R-PE-Alexa Fluor 610 496§/630

R-PE-Alexa Fluor 647 496§/668

Allophycocyanin (APC) 650/660

APC-Alexa Fluor 750 650/775

Enzymes‡

Horseradish peroxidase NA Z25054 Z25154 Z25254 Z25354

*Approximate fluorescence excitation and emission maxima, in nm.
†Each Zenon labeling kit with an Alexa Fluor dye, classic dye, or biotin contains materials for 50 labeling reactions;
one labeling reaction is defined as the amount of Zenon reagent required to label 1 μg of antibody.
‡Each Zenon labeling kit with a phycobiliprotein or enzyme contains materials for 25 labeling reactions; kits with tandem dyes contain materials for 10 labeling reactions.
§Additional excitation peaks are present at 546 and 565 nm.
NA, not applicable.
Go to thermofisher.com/flow-zenon for more information.

Secondary detection Find a small selection of conjugates in Table 9. Use our online
Secondary Antibody Selection Tool to find the right secondary
Our extensive selection of secondary detection reagents detection reagent. With this tool, you can specify target IgG
includes antibodies and streptavidin labeled with our class (and form), host, species reactivity, and conjugate type to
superior Alexa Fluor dyes, phycobiliproteins, Alexa Fluor narrow your results. Find your secondary antibody now at
dye–phycobiliprotein tandem fluorophores (Figure 19), Qdot thermofisher.com/flow-secondarydetection
labels, biotin, and enzyme labels (horseradish peroxidase
and alkaline phosphatase). We also offer antibodies with
different immunoreactivities, essential to avoid confounding
cross-reactivity when performing simultaneous secondary
immunodetection of two or more targets.

18

Table 9. Secondary detection reagent selection guide.

Dye or label

Dye Alexa Fluor Pacific Pacific Pacific Alexa PE Alexa Fluor Alexa Fluor APC Alexa Fluor
Anti-mouse IgG 405 Blue Green Orange Fluor 488 P852 610–R-PE 647–R-PE 750–APC
A865 A21006
A31553 P10993 P11204 A11001 A20980 A20990 A10931
A20981 A20991 A10540 S21008
Anti-rabbit IgG A31556 P10994 P31584 A11008 P2771MP S868,
S20982 S20992 S32362*
Anti-rat IgG A11006 A10545

Streptavidin S32351 S11222 S11200 S32365 S11223 S866

*Premium grade.

Orange uorescence
Red uorescence
Red uorescence

Green uorescence Orange uorescence Green uorescence

Figure 19. Simultaneous detection of three cell surface markers using an Alexa Fluor 610–R-phycoerythrin tandem streptavidin conjugate, Alexa Fluor 488
dye and R-phycoerythrin labels. Lymphocytes from ammonium chloride red blood cell–lysed whole blood were labeled with a biotinylated mouse anti-human
CD3 antibody, washed with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), and then incubated with Alexa Fluor 610–R-phycoerythrin
tandem dye–labeled streptavidin (Cat. No. S20982). Cells were again washed and then labeled with Alexa Fluor 488–dye conjugated anti-human CD8 antibody
and PE-conjugated anti-human CD4 antibody. After a further wash in 1% BSA/PBS, labeling was analyzed on a Becton Dickinson FACScan flow cytometer
using excitation at 488 nm. CD8 was detected in the green channel (525 + 10 nm), CD4 in the orange channel (575 + 10 nm) and CD3 in the red channel (>650
nm). The bivariate scatter plots show the expected mutually exclusive populations of (A) CD4 and CD8 positive cells, together with (B) co-positive CD3/CD4 and
(C) CD3/CD8 populations.

Custom services for flow cytometry antibodies

Our custom antibody service is efficient, and we stand behind the quality of our work. Dedicated project managers will
guide your project through every step of the process, and constantly keep you informed of our progress. Let us take the
hassle out of your hands with a custom solution for you.

We offer custom antibody services that include:
• Conjugation—using your antibody or one of ours, with the largest selection of labels available
• Formulation—including azide-free, different buffers, or different concentrations
• Packaging—bulk quantities
• Mixtures—research antibody cocktails
For more information, please email Custom Services at [email protected]

19

Cell analysis

An extensive array of Molecular Probes stains and kits have been developed to A Live Dead
assess cell function, health, and viability. Whether the health of cells is your primary
question or simply a critical factor in getting the right answers to other questions, 125 104 105
we have a solution for you. For more information and more products, go to Dead
thermofisher.com/flow-cellhealth 100
Number of cells
Cell viability 75

Cell viability assays can be used to simply distinguish between live and dead cell 50
populations, to correlate with other cell functions or treatments, or to exclude dead
cell populations from analyses. Below we describe two different cell viability assay 25
types that use only one channel of the flow cytometer, making them especially useful
with multicolor flow cytometry (Table 10). For more information and more products 0 103
go to thermofisher.com/flow-cellviability 102

LIVE/DEAD™
B

150
Live

Number of cells 100

Cell analysis LIVE/DEAD Fixable Dead Cell Stain Kits 50
• Staining retained after fixation for simple live/dead analysis with intracellular
0
phenotyping 102 103 104 105

• Fits into almost any staining and phenotyping protocol LIVE/DEAD™

• Seven colors to choose from for UV, 405, 488, or 633 lasers Figure 20. Retention of LIVE/DEAD Fixable Dead
Cell Stains after fixation. The LIVE/ DEAD Fixable
The LIVE/DEAD Fixable Dead Cell Stain Kits (see the product list, page 30) covalently Aqua Dead Cell Stain Kit (Cat. No. L34957) was
bind available amino acids but are excluded from the cytosol of live, healthy cells. used to differentially stain a mixture of live (left
The dyes react with surface proteins of both live and dead cells, but label proteins peak) and heat-treated Jurkat cells (right peak).
throughout the cytoplasm of cells with compromised membranes, causing dead Cells in (A) were not fixed; cells in (B) were fixed
cells to fluoresce at least 50 times brighter than live cells. Because the labeling is in 3.7% formaldehyde following staining. Samples
covalent, stained cells can be fixed and permeabilized without losing the viability were analyzed by flow cytometry using 405 nm
discrimination signal (Figure 20), making these reagents ideal if you want to fix and excitation and ~525 nm emission.
permeabilize samples and maintain dead-cell discrimination during subsequent
analysis. LIVE/DEAD Fixable Dead Cell Stains are available to match a range of
excitation sources and detection channels. For compensation control, use the LIVE/
DEAD Fixable Dead Cell Stains with the ArC Amine Reactive Compensation Bead Kit
(Cat. No. A10346; see page 8) for optimal results.

Table 10. Cell viability assay selection guide.

Product Target Fixable?* No-wash? Live cell Dead cell Applications
No fluorescence fluorescence Immunophenotyping
LIVE/DEAD Fixable Dead Surface and intracellular Yes Yes
Cell Stains proteins No Yes Very dim Very bright

SYTOX Dead Cell Stains Nucleic acids Very dim Very bright Live cell analyses,
Very dim Very bright dead cell exclusion
Propidium Iodide Ready Nucleic acids No
Probes Reagent Live cell analyses,
dead cell exclusion
*Formaldehyde fixation only.

20

The inadvertent inclusion of sick or dead cells in experiments AA Lymphocyte gate B Viability gate
can dramatically affect the outcomes. For instance, including CC
dead cells in immunophenotyping analysis can distort the Live cells Dead cells
results, especially for rare phenotypes. Perfetto et al. (2006)
showed that light-scatter gating during flow cytometry is FSC-A FSC-A
not enough to exclude all dead cells from analysis during
leukocyte immunophenotyping (see Figure 21 for citation). SSC LIVE/DEAD® Fixable Violet uorescence
Using LIVE/DEAD Fixable Dead Cell Stains, they were able to
efficiently exclude dead cells from analysis and, consequently, DD
significantly increase accuracy in their assays. CD4–Cy®5.5-PE uorescence CD4–Cy®5.5-PE uorescence

SYTOX Dead Cell Stains CD8–Q705 uorescence CD8–Q705 uorescence

• High-affinity nucleic acid stains for easy dead-cell Figure 21. Exclusion of dead cells eliminates staining artifacts from
discrimination analysis. After the application of a lymphocyte gate (A), live and dead cells
were discriminated (B) using LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Cat.
• Multiple colors with minimal spectral overlap for expanded No. L34955). Note the significant number of dead cells despite a scatter gate.
multicolor capabilities
Subsequent analysis of dead cells (C) and live cells (D) shows the dramatic
• No wash steps; just add, incubate, and analyze, for difference in apparent phenotypes between the two cell populations. Reprinted
simplified protocols
from Perfetto SP, Chattopadhyay PK, Lamoreaux L, et al. (2006) J Immunol
Invitrogen™ SYTOX™ Dead Cell Stains (see product list, page
30) are excluded from cells with intact membranes but Methods 313:199–208, with permission from Elsevier.
quickly diffuse into cells that have compromised membranes.
Once inside, these dyes bind DNA, which produces a A B
significant enhancement of their fluorescence; live cells remain
nonfluorescent and dead cells fluoresce brightly. SYTOX stains 1,029 1,029 105 105
are available to match widely available excitation sources.
These dyes are often used in a “dump channel,” with gating Number of events772 772 104 104
on the viable cells for further analysis (Figure 22). No wash Number of events
step is required; in fact, SYTOX Dead Cell Stains do not bind DilC1(5) uorescence515 515103 103
covalently to DNA, so dye concentrations must be maintained DilC1(5) uorescence
during analysis. 257 257

Propidium Iodide ReadyProbes Reagent 00 103 103 104 104105 105 102 102
Propidium iodide (PI) is a commonly used dead-cell stain that 100 101200 102 -102 -102
emits red fluorescence when bound to DNA. With Invitrogen™
Propidium Iodide ReadyProbes™ Reagent (Cat. No. R37108), SYTOSXY™TAOAXD™vAaAnDcevda™nceudo™resucoernecsecence 0 0 1,250 1,2502,500 2,5003,750 3,7505,500 5,500
we provide a room temperature–stable solution of the classic
DNA stain that comes in a convenient-to-use dropper bottle. RatioR3a8t3ion3m8/35n3m0 /n5m30F2nNm1F22SN12S
Just tip and drip two drops per ml to stain your cells. uoresucoernecsecexn1c,e00x01,000

Figure 22. Cell viability detected using SYTOX AADvanced Dead Cell
Stain. Jurkat cells were treated with camptothecin for 4 hours then stained with
1 µM SYTOX AADvanced Dead Cell Stain (Cat. No. S10349), 200 nM F2N12S

(Cat. No. A35137), and 50 nM MitoProbe™ DiIC1(5) (Cat. No. M34151). (A) Dead
cells were first excluded by gating live cells (those that have lower SYTOX

AADvanced fluorescence, indicated by bars). (B) Bivariant density plot shows
a two-parameter apoptosis assay for mitochondrial membrane potential loss

(decreased DiIC1(5) fluorescence) and breakdown of membrane asymmetry

(smaller F2N12S 585/530 nm fluorescence ratio). The A, L, and D labels on the
graphs indicate apoptotic, live, and dead cells, respectively. Control data are

not shown.

21

Cell proliferation

Cell proliferation and the characterization of agents that either promote or retard A 200 Number of cells Number of cells 900
cell proliferation are extremely important areas of cell biology and drug discovery 800
research. We offer both traditional reagents for assessing cell proliferation 150 700
(CellTrace Cell Proliferation Kits), as well as the latest technology for measuring 100 600
new DNA synthesis (Click-iT Plus EdU labeling) (Table 11). For more information 500
and more products, go to thermofisher.com/flow-cellproliferation 50 400
300
Click-iT Plus EdU Flow Cytometry Assay Kits 0 200
101 102 103 104 105 106 100

• Superior accuracy compared to BrdU assays, with minimal variation (low CV Click-iT™ EdU plus Alexa Fluor™ 488 uorescence 0
values) 0.0
B 900
• Streamlined five-step protocol 200 Click-iT™ EdU plus FxC
800 Alexa Fluor™ 488 uorescence
106
• Multiplexable with GFP, mCherry, APC, PerCP, PE, a15n0 d other fluorophores Number of cells Number of cells 700
600 105

100 500 104
400
The growth of cells within a population can be indirectly observed by measuring 300 103
modified nucleoside incorporation into newly synthesize50d DNA. EdU (5-ethynyl- 200
102
2´-deoxyuridine) is a nucleoside analog that is incorporated into DNA during 100 0.0
0 0
0.0 0.5 1.0 1.5 2.0 F
synthesis. The Invitrogen™ Click-iT™ Plus EdU Flow Cytom10e1 try10A2 ssa10y3 Kit1s04allo1w05 106
detection of EdU (which contains an alkyne) by a coppCleicrk--ciTa™tEadlUyzpeluds ArleexaacFtluioorn™ 4th88atuorescence FxCycle™ Violet uorescence (x106)

produces a stable covalent bond between the alkyne and a fluorescent dye– C
az20i0de. 900
labeled picolyl The small size of the picolyl reagents means 106Click-iT™ EdU plus
azide800detection Alexa Fluor™ 488 uorescence
105
that the fluoresc1e50nt label has efficient access to the in7t0a0ct DNA without the need
Cell analysis Number of cells 600 104
Number of cellsfor harsh cell treatment.
500 103

100 400 102
0.0 0.5 1.0 1.5 2.0
In the past, DNA synthesis was measured by incorpor3a00ting the nucleoside analog
bromodeoxyuridi5n0e (BrdU) into DNA, followed by dete2c00tion with an anti-BrdU FxCycle™ Violet uorescence (x106)
100
antibody. Althoug0h useful in its time, that method requir0es DNA denaturation Figure 23. Cell proliferation analysis using
heaCtl,icok10-ri1TD™ EN1d0Ua2 spelu)s10tA3olexeax1F0pl4uoors™e14085t8heuo1B0re6rsdceUncteo a0.n0FtxibCyocdl0e.y5™—Vioalets1.tu0eorpestchen1a.c5te 2.0 the Click-iT Plus EdU Alexa Fluor 488 Flow
(using acid, the Cytometry Assay Kit and FxCycle Violet Stain.
(x106) Jurkat cells were treated with 10 μM EdU for one
hour and stained with Alexa Fluor 488 picolyl azide,
can adversely affect sample quality. The Click-iT Plus EdU Flow Cytometry Assay according to the Click-iT Plus EdU Alexa Fluor
488 Flow Cytometry Assay Kit’s protocol (Cat.
eliminates the need to denature DNA, thus simplifying the assay considerably yet No. C10632), followed by staining with FxCycle
Violet Stain (Cat. No. F10347). Cells were then
generating comparable results (Figure 23). Click-iT Plus EdU labeling is compatible analyzed by flow cytometry using either 488 nm
excitation (for Click-iT EdU Alexa Fluor 488 dye)
with fixation protocols. or 405 nm excitation (for FxCycle Violet Stain).
(A) Histogram demonstrating clear separation of
The Click-iT Plus kits can be used with fluorescent proteins, because the Click-iT cells in S phase (DNA synthesis, including EdU
Plus reaction uses a modified azide in place of the azide used in the original Click- incorporation) and cells in either G2/M or G0/G1.
iT reaction. As a result of the modification, the concentration of free copper in the (B) Histogram showing DNA content distribution,
sample is significantly lower and fluorescence signals from fluorescent proteins with G0/G1 and G2/M phase peaks separated by
(e.g., R-PE, R-PE tandem dyes, and GFP) are not quenched. The speed and the S phase distribution using FxCycle Violet Stain.
accuracy of the Click-iT Plus EdU reaction is comparable to that of the original (C) Dual-parameter Click-iT Plus EdU and FxCycle
Click-iT EdU reaction. See the product list on page 30 for available kits. plot shows co-positive cells that provide a direct
measurement of the percentage of cells in S phase.

22

Table 11. Cell proliferation assay selection guide.

Product Target Fixable Multiplexing Application

Click-iT Plus EdU Flow Cytometry Assay Kits Incorporation into newly synthesized DNA Yes Yes Cell proliferation
CellTrace Cell Proliferation Kits Lysine-containing proteins Yes Yes Generational analysis

CellTrace Cell Proliferation Kits

• Bright single-peak staining enables visualization of couple to cellular proteins by reaction with lysine side chains
multiple generations and other available amines. When cells divide, the CellTrace

• Well retained in cells for several days post-staining dye labeling is distributed equally between the daughter
cells, and each successive generation in a population
• No known cytotoxic effect on proliferative ability or of proliferating cells is marked by a halving of cellular
biology of cells fluorescence intensity. Eight to ten successive generations

• Multiple colors available to easily combine with antibodies have been identified with both CellTrace CFSE and CellTrace
or markers of cell function, such as GFP Violet dye (Figure1020 4). All of the CellTrace dyes can be used
in combination with other cell function probes or markers.
4000
80
• Simple, robust staining protocol
Because CellTrace Violet and CellTrace Far Red use lasers
3000 Cell count
Cell count
The Invitrogen™ CellTrace™ family of dyes comprises and detection cha60nnels different from those used for green
2000 fluorescence, mu4lt0iplexing with GFP, fluorescein, or other

CellTrace CFSE dye (Cat. No. C34554), CellTrace Violet

dye (Cat. No. C34557), and CellTrace Far R10e0d0 dye (Cat. green-fluorescent20probes is possible.

No. C34564), all of which spontaneously and irreversibly 105 106 0 106
0 103 104 105
102 103 104

CellTrace™ Violet uorescence CellTrace™ CFSE uorescence

A BC

Cell count 40004000 Cell count 100 100 100 Cell count 100
Cell count30003000 Cell count 80 80 80 80
20002000 Cell count 60 60 60 60
10001000 40 40 40 40
20 20 20 20

00 000 104 11004 4 10511004 5 106 106 0
102 102 103 103 104 104 105 105 106 106 102 103 103 103 103 104 105 106

CellCTreallcTera™cVei™olVeitoleutoreusocresncceence CelClTerallCcTrea™llcTYerae™cllCeo™wFSCEFuSourEeosrceuesoncrecsencceence CellTrace™ Far Red uorescence

Figure 24. Generational tracing using CellTrace reagents. Cell proliferation was followed for varying numbers of generations under different experimental

conditions u10s0in10g0 (A) CellTrace Violet reagent in human per1i0p0h1e00ral blood mononuclear cells (PBMCs) stimulated for 7 days; (B) CellTrace CFSE reagent in

human T lymphocytes stimulated for 5 days; and (C) CellTrace Far Red reagent in human T lymphocytes stimulated for 5 days. PBMCs were stimulated

with anti-hum80an80CD3 antibody (Cat. No. MHCD0300) and i8n0te8r0leukin-2 (Cat. No. PHC0027) while human T lymphocytes were stimulated with anti-human
CD3 antibody (Cat. No. MHCD0300) alone. Unstimulated parent generations are represented by (A) red peak; (B) blue peak and (C) purple peak. Dead cells
Cell count Cell count
were exclud6e0d 6f0rom each data group using SYTOX Dead C6e0ll 6S0tains. Analyses were performed using an Attune flow cytometer with the following excitation:Cell countCell count
bandpass emission filter for each reagent: 405 nm: 450/40 nm for CellTrace Violet detection; 488 nm: 530/30 nm for CellTrace CFSE detection; and 638 nm:

660/20 nm f4o0r C40ellTrace Far Red detection. 40 40

20 20 20 20

00 103 103 104 104 104 104 00 104 104 105 105 106 106
102 102 103 103

CellCTreallcTera™cYee™llYoewllowuoreusocresncceence CellCTreallcTera™cFea™rFRaerdReudoreusocresncceence

23

Cell cycle

Detection of DNA content provides a snapshot of cells in a FxCycle Stains
population that are in different stages of the cell cycle. Flow • Allows increased flexibility for multicolor cell cycle studies
cytometry, in conjunction with modeling algorithms, provides
a powerful tool to assess cells in G0/G1 phase versus S • Requires little or no compensation with 488 nm excitable
phase, G2/M phase, or showing polyploidy. Molecular Probes dyes
fluorescent dyes allow accurate cell cycle analysis in either live
or fixed cell populations (Table 12). For more information and to • Tight CV values enable more accurate analysis
see more products, go to thermofisher.com/flow-cellcycle
Cell cycle analysis with Invitrogen™ FxCycle™ Violet Stain
Vybrant DyeCycle Stains (Cat. No. F10347), FxCycle PI/RNase Staining Solution
• Enables accurate cell cycle analysis in living cells (Cat. No. F10797), or FxCycle Far Red Stain (Cat. No.
F10348) allows easy multiplexing with other cellular assays,
• Low cytotoxicity for cell sorting and additional live cell such as those measuring cell proliferation (Figure 23, page
experiments 22), immunophenotype, apoptosis, or viability, by freeing up
other lasers and detection channels. The FxCycle stains,
• Multiple color options for easier multiplexing intended for cells that are fixed and permeabilized, provide
fluorescence signals proportional to the DNA content of
• Ability to sort based on phase of cell cycle each cell in a population.

• Ability to identify stem cell side populations using the A B
violet laser
105 2,000
Invitrogen™ Vybrant™ DyeCycle™ stains offer the ability to
stain for DNA profiling in live cells, with options for 405, 488,
532, or 633 nm excitation. The dyes are generally used in
combination with a dead-cell stain (Figure 25) to exclude
dead cells from the analysis, but the Vybrant DyeCycle stains
are not cytotoxic, allowing stained cells to be sorted and then
cultured or assessed with functional assays after determining
their cell cycle stage. See the product list on page 30 for the
available reagents.
Cell analysis 104 1,500
SYTOX™ Blue uorescenceDead
Number of cells
1,000
103

102 500
Live
0
0 50 100 150 200 250 0 50 100 150 200 250

Vybrant™ DyeCycle™ Green uorescence Vybrant™ DyeCycle™ Green uorescence

Figure 25. Viable-cell gating with Vybrant DyeCycle Stains. Jurkat cells from
an overgrown culture were stained with Vybrant DyeCycle Green Stain (Cat. No.
V35004) and then SYTOX Blue Dead Cell Stain (Cat. No. S34857) and analyzed
by flow cytometry using 488 nm and 405 nm excitation. The histogram (B) was
gated on live cells (A) and shows DNA content distribution in live cells: G0/G1
and G2/M phase peaks are separated by the S phase distribution. Inclusion of
the dead cells would have produced aberrant results.

Table 12. Cell cycle assay selection guide.

Product Name Target Live cells? Multiplexing? Application
Yes Yes Live cell analysis
Vybrant DyeCycle Stains DNA No Yes Fixed cell analysis

FxCycle Stains DNA

24

Apoptosis

Apoptosis is distinct from necrosis in both the biochemical and the A 106 Induce

morphological changes that occur. In contrast to necrotic cells, apoptotic cells 106 Control N 105

are characterized morphologically by compaction of the nuclear chromatin, 105SYTOX™ AADvanced™ SYTOX™ AADvanced™ 104

shrinkage of the cytoplasm, and production of membrane-bound apoptotic 104

bodies. Biochemically, apoptosis is distinguished by fragmentation of the

genome and cleavage or degradation of several cellular proteins. As with cell 102 102
–103 –103

viability, no single parameter fully defines cell death in all systems; therefore, VA V

it is often advantageous to use several different approaches when studying –104 103 105 105 –104
–103 –102 –103 –102
apoptosis. Below we describe several methods and Molecular Probes products
CellEvent™ Caspase-3/7 Green uorescence CellEvent™

for assessing apoptosis (Table 13). For more information and more products for

apoptosis, go to thermofisher.com/flow-apoptosis Control N B N
106
106 Induced
105SYTOX™ AADvanced™ SYTOX™ AADvanced™
105
CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit
104 104

• Optimized caspase-3/7 substrate for apoptosis analysis

• Simple, no-wash protocol helps preserve delicate 102 102
–103 –103
apoptotic cells
V A VA

• Compatible with both live-cell fluorescence imaging–10a4 nd formaldehyde-based –104
–103 –102 103 105 105 –103 –102 103 105 105
fixation methods
CellEvent™ Caspase-3/7 Green uorescence CellEvent™ Caspase-3/7 Green uorescence

Caspases are a family of enzymes that play key roles in initiating and effecting Figure 26. Detection of caspase activity in
apoptosis, and activation of caspase enzymes is a distinctive feature of the early
stages of apoptosis. We provide a variety of caspase assays for flow cytometry, Jurkat cells, using the CellEvent Caspase-3/7
including the Invitrogen™ CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Green Flow Cytometry Assay Kit. Jurkat cells
Kit (Cat. No. C10427), which detects caspase activity with a substrate that, after (human T cell leukemia) were treated with (A)
being cleaved by caspase-3 or caspase-7, binds to DNA and becomes brightly DMSO or (B) 10 μM camptothecin for 3 hours
fluorescent (Figure 26). before labeling with the CellEvent Caspase-3/7
Green Flow Cytometry Assay Kit (Cat. No.
C10427). Stained samples were analyzed on the
Attune flow cytometer equipped with a 488 nm
laser, and fluorescence emission was collected
using a 530/30 nm bandpass filter for the
CellEvent reagent and a 690/50 nm bandpass
filter for the SYTOX AADvanced stain (also
provided in the kit). Note that treated cells have
a higher percentage of apoptotic cells (B) than
the basal level of apoptosis seen in the control
cells (A). A = apoptotic cells; N = necrotic cells;
V = viable cells.

Table 13. Apoptosis assay selection guide.

Product name Target Fixable Multiplexing No. of channels used
Caspase 3/7 No Yes 1
CellEvent Caspase-3/7 Green
Flow Cytometry Assay Kit Phosphatidylserine translocation Yes Yes 1
No Yes 2
Annexin V conjugates No Yes 3

MitoProbe JC-1 Assay Kit Mitochondrial membrane potential

Violet Ratiometric Membrane Asymmetry Asymmetric membranes
Probe/Dead Cell Apoptosis Kit

25

Annexin V conjugates and kits MitoProbe JC-1 Assay Kit

• Conjugated to Molecular Probes dyes for increased • Easy to use and compatible with existing research
sensitivity protocols

• Conjugates for all available lasers for increased • Can be used with multiple cell types
multiplexing capabilities
• Available for 488 nm and 633/635 nm excitation
• Available as stand-alone reagents or easy-to-use kits
• Low compensation alternatives
Another hallmark of apoptosis is the translocation of
phosphatidylserine (PS) from the cytoplasmic surface of cell The Invitrogen™ MitoProbe™ JC-1 Assay Kit (Cat. No.
membranes, where it normally resides, to the external surface M34152) provides the cationic dye JC-1, carbonyl
of cells. Once this translocation has occurred in apoptotic cyanide 3-chlorophenylhydrazone (CCCP, a mitochondrial
cells, the PS can be detected by the binding of fluorescently- membrane–potential uncoupler), dimethylsulfoxide (DMSO),
labeled annexin V, a PS-binding protein. We offer an array of and concentrated phosphate-buffered saline (PBS) for the
fluorescent conjugates of annexin V, including conjugates of study of mitochondrial membrane potential. JC-1 exhibits
Molecular Probes bright and photostable Alexa Fluor dyes potential-dependent accumulation in mitochondria, indicated
(Figure 27) and an Annexin-binding Buffer (Cat. No. V13246) by a fluorescence emission shift from green (~529 nm) to
designed to facilitate PS binding. Additionally, we have flow red (~590 nm), due to concentration-dependent formation
cytometry–optimized kits that combine some of our other of red-fluorescent J-aggregates (Figure 28). Consequently,
viability and cell function probes with annexin to provide mitochondrial depolarization is indicated by a decrease in the
multiparametric data on the apoptotic state of cells. Three red-to-green fluorescence intensity ratio, which is dependent
of those kits and several annexin V fluorescent conjugates only on the membrane potential and not on other factors
are featured in the product list on page 31. To find all of the such as mitochondrial size, shape, or density, which may
available apoptosis kits, go to thermofisher.com/flow-annexin influence single-component fluorescence measurements. Use
of fluorescence ratio detection therefore allows researchers to
A B make comparative measurements of membrane potential and
to determine the percentage of cells within a population that
110044 110044 respond to an applied stimulus.
Cell analysis DD DD
110033 110033
PPrrooppiiddiiuumm iiooddiiddee uuoorreesscceennccee
PPrrooppiiddiiuumm iiooddiiddee uuoorreesscceennccee110022110022A Untreated B
JC-1 red uorescence - BL-3VVAA VV
JC-1JreC-d1 rueodrfleusocreenscceen-cBeL-3AA Treated
JC-J1 rCe-d1 reudorfleusocresncen-cBeL-3110011110011
JC-1 red uorescence - BL-3
110000 110011 110022 110033 110044 110000 110011 110022 110033 110044
110000 110000

AAlelexxaaFFluluoorr™™448888 uuoorreesscceennccee AAlelexxaaFFluluoorr™™448888 uuoorreesscceennccee

Figure 27. Flow cytometric analysis of Jurkat cells using the Alexa Fluor JC-J1Cg-1reJgCern-e1egnuroereuenosrcfeleusnoccreensc- eBn-Lc1Be-LH1-H JC-J1CJg-C1re-ge1rnegerenueonrueflosurcoeersencscecenec-necBe-L1B-LH1-H
488 annexin V conjugate and propidium iodide. Jurkat human T cell
leukemia cells were first exposed to 10 μM camptothecin for four hours Figure 28. Jurkat cells stained with 2 μM JC-1. Cells were stained using
(A) or left untreated (as control, B). Cells were then treated with the reagents
in the Invitrogen™ Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & reagents in the MitoProbe JC-1 Assay Kit (Cat. No. M34152) for 20 minutes
Propidium Iodide (Cat. No. V13245) and analyzed by flow cytometry. Note
that the camptothecin-treated cells have a significantly higher percentage
of apoptotic cells (labeled “A”) than the basal level of apoptosis seen in the
control cells. V = viable cells; D = dead cells.

at 37°C and 5% C48O82, washed with PBS, and analyzed on the Attune flow
cy tometer using nm excitation with 530/30 nm bandpass and >640 nm

longpass emission filters. Untreated cultured cells (A) are shown compared

to treated cells (B), which were induced to undergo apoptosis with 10 μM

camptothecin for 5 hours at 37°C.

26

Membrane asymmetry probe potential. The dye exhibits an excited-state intramolecular
• Enables accurate apoptotic analysis on trypsinized cells proton transfer reaction resulting in dual fluorescence, with
two emission bands corresponding to 530 nm and 585 nm,
• Simple 5-minute staining protocol producing a two-color ratiometric response to variations in
surface charge. The F2N12S probe is combined with SYTOX
• Compatible with other blue-excited apoptotic stains AADvanced dead cell stain, which is capable of passing
through the cell membrane only in late apoptotic or necrotic
The Invitrogen™ Violet Ratiometric Membrane Asymmetry cells, allowing discrimination from early apoptotic cells.
Probe/Dead Cell Apoptosis Kit (Cat. No. A35137) provides
an easy, efficient method for the detection of apoptosis with Unlike annexin-based assays, this assay does not require
dead-cell discrimination using a violet laser flow cytometer special buffers or wash steps, and it is less susceptible to the
(Figure 29). The Violet Ratiometric Membrane Asymmetry cell membrane damage commonly found during the physical
Probe detects the membrane asymmetry changes during or chemical removal steps when assaying adherent cells,
apoptosis. It works well on adherent and suspension cells, therefore providing better data quality.
and correlates with other indicators of apoptosis, such as
caspase detection and changes in mitochondrial membrane

A B

262,144 262,144

F2N12S green uorescence 196,608 A+D F2N12S green uorescence 196,608 A+D
131,072 131,072

65,536 L 65,536 L
0 0
0 65,536 131,072 196,608 262,144 0 65,536 131,072 196,608 262,144

F2N12S orange uorescence F2N12S orange uorescence

C 105 SYTOX® AADvanced™ uorescence D D 105 SYTOX® AADvanced™ uorescence D

104 104

103 A L 103 L
102 A

102

101 1,250 2,500 3,750 5,000 101 1,250 2,500 3,750 5,000
0 0

F2N12S ratio 585 nm/530 nm F2N12S ratio 585 nm/530 nm
uorescence x 1,000 uorescence x 1,000

Figure 29. Violet ratiometric membrane asymmetry probe for apoptosis detection. Jurkat cells (T cell leukemia, human) were treated with 10 μM

camptothecin for 4 hours (panels B and D) or left untreated as a control (panels A and C). Samples were analyzed on a flow cytometer with 405 nm excitation
using 585 nm and 530 nm bandpass filters for F2N12S, and 488 nm excitation for SYTOX AADvanced dead cell stain using a 695 nm bandpass filter. Live cells

can be discriminated from apoptotic and dead cells by the relative intensities of the two emission bands from F2N12S (A and B). In panels C and D, SYTOX
AADvanced dead cell stain fluorescence is plotted against a derived ratio parameter from the two emission bands (585/530 nm) of F2N12S. A = apoptotic cells,
L = live cells, D = dead cells.

27

Other cell function assays

CellROX Flow Cytometry Kits Invitrogen™ CellROX™ reagents are fluorogenic probes
for measuring generalized oxidative stress in cells, using
• Fluorogenic probe that is oxidized to a fluorescent form in conventional fluorescence microscopy, high-content
the presence of reactive oxygen species (ROS) screening, microplate fluorometry, or flow cytometry (Figure
30). In a reduced state, the dyes are nonfluorescent, but
• Minimal overlap with fluorophores excited by other laser upon oxidation, they fluoresce brightly. Molecular Probes kits
lines, allowing easy multiplexing for flow cytometry include CellROX Green Flow Cytometry
Assay Kit (Cat. No. C10492), CellROX Orange Flow
• Cells can be stained in complete media or other Cytometry Assay Kit (Cat. No. C10493), and CellROX Deep
appropriate buffer; no need for serum-free media Red Flow Cytometry Assay Kit (Cat. No. C10491). For more
information, go to thermofisher.com/flow-cellrox
Generation of ROS is inevitable for aerobic organisms
and, in healthy cells, occurs at a controlled rate. Under
conditions of oxidative stress, ROS production is dramatically
increased, resulting in subsequent alteration of membrane
lipids, proteins, and nucleic acids. Oxidative damage of
these biomolecules is associated with aging, as well as with
a variety of pathological events, including atherosclerosis,
carcinogenesis, ischemia-reperfusion injury, and
neurodegenerative disorders.

AB C

105 105
Cell analysis

Number of cells
SYTOX™ Blue uorescence
SYTOX™ Blue uorescence
104 104

103 103

102 102
00

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105

CellROX™ Deep Red uorescence CellROX™ Deep Red uorescence CellROX™ Deep Red uorescence

Figure 30. ROS detection by flow cytometry. (A) ROS levels detected by the CellROX Deep Red Reagent (provided in the CellROX Deep Red Flow Cytometry
Assay Kit, (Cat. No. C10491) are decreased in oxidant-treated Jurkat cells with pretreatment of cultures using N-acetylcysteine (NAC). The cells treated with the

oxidant tert-butyl hydroperoxide (TBHP) (red) show increased staining with the CellROX Deep Red Reagent compared with cells pretreated with NAC before

TBHP treatment (blue) and with untreated control cells (green). (B, C) CellROX Deep Red Reagent can be used with SYTOX Blue Dead Cell Stain to differentiate
live stressed cells from dead cells. Jurkat cells were treated with (B) PBS or (C) 200 μM TBHP for 30 minutes before labeling using the CellROX Deep Red Flow
Cytometry Assay Kit. Note that the treated cells (C) have a higher percentage of cells under oxidative stress than the basal level of ROS observed in control cells (B).

28

pHrodo E. coli BioParticles Phagocytosis Kits Number of cells counted 120
• Specifically detect and monitor phagocytosis and
Negative
endocytosis control
80
• Track antibody internalization with pHrodo conjugates
Phagocytosed
• Track ligand internalization with pHrodo conjugates particles

• Combine with red or green dyes for multiplexed 40
experiments

Proprietary, pH-sensitive Molecular Probes pHrodo dyes are 0 101 102 103 104
almost nonfluorescent at neutral pH and fluoresce brightly 100
in acidic environments, making them ideal for use as pH
indicators for a variety of applications, including studying the pHrodo™ dye uorescence (585 nm)
processes of phagocytosis and endocytosis. The Invitrogen™
pHrodo™ Green E. coli BioParticles™ Phagocytosis Kit Figure 31. Flow cytometry analysis showing increased fluorescence of
(Cat. No. P35381) and the Invitrogen pHrodo Red E. coli granulocytes treated with pHrodo Red BioParticles conjugates. A whole
BioParticles Phagocytosis Kit (Cat. No. A10025; Figure 31) blood sample was collected and treated with heparin, and two 100 µL aliquots
enable the detection of phagocytic activity in whole blood were prepared. Both aliquots were treated with pHrodo Red E. coli BioParticles
samples and live cell lines by flow cytometry. The kits include conjugates (used in the pHrodo Red E. coli BioParticles Phagocytosis Kit, Cat.
all of the reagents required for assessing particle ingestion No. A10025) and vortexed. One sample was placed in a 37°C water bath, and
and red blood cell lysis. Additionally, the Invitrogen pHrodo the other sample (negative control) was placed in an ice bath. After a 15-minute
Red Phagocytosis Particle Labeling Kit (Cat. No. A10026) incubation, red blood cells were lysed with an ammonium chloride–based lysis
allows you to label your own sample of bacteria with pHrodo buffer. The samples were centrifuged for 5 minutes at 500 x g, washed once,
Red dye. To find more information and products, go to and resuspended in Hank's Balanced Salt Solution. The samples were then
thermofisher.com/flow-phrodo analyzed on a Becton Dickinson FACSCalibur™ cytometer using a 488 nm argon
laser and 564–606 nm emission filter. The sample incubated at 37°C shows
the increased fluorescence of the phagocytosed pHrodo Red BioParticles
conjugates (red), in contrast to the negative control sample, which was kept on
ice to inhibit phagocytosis (blue).

29