MP Flow Cytometry Resource Guide

Molecular Probes flow cytometry
Products and resource guide
Beautiful data again and again

Resources
Reference guide
The Molecular Probes Handbook, 11th Edition
®
The most complete reference on fluorescent labeling and detection available, this resource features extensive references and technical notes, and
contains over 3,000 technology solutions representing a wide range of biomolecular labeling and detection reagents. See the online version of
The Molecular Probes Handbook and request your free copy* at lifetechnologies.com/handbook
®
Online tools Stay in the know—news and updates

Antibody selection tool BioProbes Journal and
®
Explore our extensive portfolio of high-quality primary and Molecular Probes newsletter
®
secondary antibody conjugates with the easy-to-use selection Our award-winning print and online magazine,
tool. lifetechnologies.com/antibodies BioProbes Journal, highlights the latest
®
breakthroughs from our scientists, featuring new

technologies and products.
Fluorescence SpectraViewer (online)
Plot up to 14 fluorophores on a single graph, which you can The Molecular Probes newsletter keeps you informed
®
then print or save for later. lifetechnologies.com/spectraviewer about new products that have just been released.
To access current and past issues of the BioProbes ®
Journal, go to lifetechnologies.com/bioprobes
Flow cytometry resources center To receive the BioProbes Journal and the Molecular Probes newsletter,
® ®
Search for protocols, tutorials, application notes, fluorophore and subscribe at lifetechnologies.com/subscribeprobes
product selection guides, literature, and many other technical
resources in a single place. lifetechnologies.com/flowresources
Learn and connect
Flow cytometers and accessories Molecular Probes webinar series
®
The next step in the evolution of cell-by-cell analysis is here. Our scientists share various techniques
The Attune NxT Acoustic Focusing Cytometer enables you
®
and provide tips and tricks.
to optimize performance and throughput without sacrificing
sensitivity or accuracy. lifetechnologies.com/attune View the recorded webinars or
sign up for future live webinars at
lifetechnologies.com/probeswebinars
Mobile apps for research on the go

Flow cytometry guide and protocols
Get access to our reagent selection guides for selected @facebook.com/LifeTechnologies
applications and protocols, and troubleshoot on the go. Also @facebook.com/LIFE/MolecularProbes
enjoy a built-in protocol timer and alarm for timed steps.
Fluorescence SpectraViewer @twitter.com/LIFECorporation

Plot and compare spectra, check spectral compatibility for @twitter.com/molprobes
fluorophores, and email the configuration to yourself in a clear,
printable format.
@linkedin.com/lifetechnologies
DailyCalcs science calculator @youtube.com/lifetechnologies
Easily calculate molarity, dilution, molecular weight, and more.
All the mobile apps are available free of charge, and can be downloaded at
lifetechnologies.com/apps
*Not available in all countries.
Find out more at lifetechnologies.com/probes
Molecular Probes® | Flow cytometry products

Molecular Probes flow cytometry ®
Products and resource guide

Flow cytometry workflow
The Molecular Probes flow cytometry products and resource
®
guide presents an overview of primary antibody conjugates, cell

function assays, and other tools optimized for flow cytometry
by our scientists. The Molecular Probes team has been at the
forefront of invention and development of fluorescent probes for
nearly 40 years, and this guide features some of the most useful
flow cytometry tools available anywhere. For the probes and
assays you need, available in a full spectrum of fluorescent colors,
look to Molecular Probes flow cytometry products first.
®
Visit lifetechnologies.com/flow-cytometry for more information Antigen detection

on Molecular Probes flow cytometry products and resources.
®
Primary antibodies ___________ 12
Fluorophore overview _________ 12
Antibody labeling _____________ 15
Secondary detection __________ 18
Custom services_____________ 19
Antigen detection
Instrument set-up
Sample preparation
and calibration
Sample preparation Instrument set-up Cell analysis

Cell preservation ______________ 2 and calibration
Red blood cell lysis____________ 2 Alignment ___________________ 6
Fixation and permeabilization ___ 2 Size calibration _______________ 6
Dynabeads cell isolation_______
®
3 Cell sorting set-up ____________ 7 Cell analysis
Compensation ________________ 7 Viability ____________________ 20
Absolute cell counting _________ 9 Cell proliferation _____________ 22
Cell cycle ___________________ 24
Apoptosis ___________________ 25
Other cell function assays _____ 28
Molecular Probes® | Flow cytometry products

Sample preparation
Quality data require quality starting material. Molecular Probes
®
The FIX & PERM Cell Permeabilization Kit (Cat. No. GAS003,
®
sample preparation reagents, which include reagents for blood GAS004) consists of matched Fixation Reagent (Medium A) and
cell preservation, red blood cell lysis, and sample fixation and Permeabilization Reagent (Medium B) for simultaneous analysis
permeabilization, are designed to help you achieve the best of intracellular and cell-surface antigens in the same cell
possible results. Learn more about these products and get population (Figure 1). This procedure facilitates antibody access
protocols at lifetechnologies.com/flow-sample to intracellular structures and leaves the morphological light-
scattering characteristics of the cells intact. These formulations
reduce background staining and allow simultaneous addition
Cell preservation of permeabilization medium and fluorophore-labeled
Sample preparation
antibodies. The Fixation Medium (Cat. No. GAS001S100) and

TransFix Reagent
®
Permeabilization Medium (Cat. No. GAS002S100) are available
TransFix Reagent (Cat. Nos. FIX2, FIX20, FIX100) is a whole-
®
separately as well.
blood preservative that stabilizes leukocytes and preserves
antigenic sites for future cellular analysis. The reagent allows
simple batch processing of multiple samples to optimize your A
laboratory workflow. Additionally, blood that has been stabilized
TNF-α–Pacific Blue™ fluorescence

with TransFix Reagent can be easily transported to other sites
® 106 106
for analysis. 10 5
105
Cell lysis 104 104
Cal-Lyse Whole Blood Lysing Solution

™
103 103
-103 -103
The Cal-Lyse premixed lysing solution (Cat. Nos. GAS010,
™ -103 103 104 105 106 -103 103 104 105 106
γ-IFN–PE-Cy®7 fluorescence γ-IFN–PE-Cy®7 fluorescence
GAS010S100) is specifically formulated to lyse red blood cells B
following monoclonal antibody staining of whole blood or bone
γ-IFN–PE-Cy®7 fluorescence
marrow. Treatment with this reagent leads to both the lysis of 106 106
red blood cells and the fixation of white cells. Treatment does 10 5
105
not affect fluorophore-labeled antibodies bound to leukocytes,

and leaves morphological light-scattering characteristics of
leukocytes intact. Cal-Lyse reagent can be used for either “no-
™
104 104
wash” or “wash” staining procedures.

103 103
-103 -103
Fixative-Free High-Yield Lyse -103 103 104

CD4–Fluorescein fluorescence
105 106 -103 103 104 105 106
CD4–Fluorescein fluorescence
High-Yield Lyse (Cat. No. HYL250) is a premixed, fixative-free

erythrocyte lysing solution that can be used to eliminate red cells Figure 1. Use of FIX & PERM Cell Permeabilization Kit for simultaneous
®
surface antigen and intracellular antigen staining. C57BL/6 splenocytes were

from whole blood for flow cytometric analysis, with minimal loss left unstimulated or stimulated for 5 hours with phorbol myristate acetate
of rare blood cell populations. Using this reagent, erythrocyte (PMA) and ionomycin in the presence of brefeldin A. Cells were then surface-
lysis is performed immediately following staining of the blood stained with fluorescein (FITC)-conjugated anti–mouse CD4 antibody
samples with monoclonal antibodies; therefore, there is no need (Cat. No. MCD0401). This step was followed by fixation and permeabilization of
for a wash step. the sample using the FIX & PERM Cell Permeabilization Kit (Cat. No. GAS003, ®
GAS004). Intracellular staining was performed during the permeabilization

step using PE-Cy 7 tandem–conjugated anti–mouse γ-interferon (γ-IFN)
®
Fixation and permeabilization antibody (Cat. No. A18713) and Pacific Blue dye–conjugated anti–mouse ™
tumor necrosis factor α (TNF-α) antibody (Cat. No. RM90128). Data were
collected using the Attune Acoustic Focusing Cytometer (blue/violet)
®
FIX & PERM reagents and kits

®
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-Cy 7 tandem ®
• Compatible with analysis of most cellular antigens fluorescence. Pacific Blue conjugate fluorescence was detected using 405
™
nm excitation and a 450/40 nm bandpass emission filter. (A) γ-IFN and TNF-α
• No effect on cellular morphological scatter
antibody co-staining of total mouse splenocytes, gated on lymphocytes, that
• Reduced background staining were left unstimulated (left) or stimulated (right) with PMA and ionomycin
in the presence of brefeldin A. (B) CD4+ T cell expression of TNF-α (left) and
• Proven protocols γ-IFN (right) after stimulation as described above.
2 Molecular Probes® | Flow cytometry products

Dynabeads cell isolation
®
• Viable and functional—products for positive isolation, negative isolation, cell

activation, and depletion Incubate your
starting sample
• Gentle—column-free separation and inert bead surfaces translate to gentler with Dynabeads®
handling of your cells and reduce the risk of contaminants in the preparations
• High yields—tube-based separation allows you to achieve excellent recovery of cells
Sample preparation
Separate
bead-captured
When cells are removed from their natural environment, there is a risk that cells with a
Dynal MPC™
magnet
experimental procedures will negatively impact cell phenotype and function. Choosing
the right cell separation method is therefore critical to downstream experiments.
Dynabeads magnetic beads are superparamagnetic, monosized polymer beads coated
®
with a thin, inert polymer shell to encase the magnetic material (Figure 2). This design
helps to eliminate the risk that any unwanted material such as iron is left in the sample Transfer
after separation. In addition, since the cells are never bound to the beads (negative- supernatant with
untouched cells
to a new tube
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).
Negatively Positively
isolated cells isolated cells
(untouched) (bead-captured)
Human cell isolation 1
2 ads
ove be
Dynabeads magnetic beads enable gentle tube-based isolation of human cells directly
® Rem 3
Cell-based assays, flow Molecular applications

from whole blood, mononuclear cells, buffy coat, bone marrow, or tissue samples for any cytometry, cell expansion e.g., mRNA and DNA analysis
downstream assay, including flow cytometry (Figure 4). Dynabeads products are available
®
Figure 3. Workflow diagram for using Dynabeads ®
for isolating human T cells, B cells, stem cells, NK cells, monocytes, dendritic cells, beads for positive or negative tube-based cell
endothelial cells, tumor cells, leukocytes, and granulocytes. isolation.
Isolation options include:

1,000 1,250
• Positive isolation and cell release
2,000
• Negative isolation, resulting in untouched cells
1,500
• Depletion of unwanted cell types or positive cell isolation for
750
Count
Count
1,000
molecular applications
500
• Isolation of cells using your own antibody
500
0250
If you can’t find a ready-to-use product for human cell isolation, we have a range of 0
102 103 104 105 102 103 10
Dynabeads products that can be combined with an antibody of your choice to create a
®
CD4 FITC CD4 FITC
tailored cell isolation tool that include: streptavidin Dynabeads , secondary antibody–
®
1,000 1,250
2,000
coated Dynabeads , and surface-activated Dynabeads . Learn more at

® ®
lifetechnologies.com/humancellisolation
1,500
750
Count
Count
1,000
500
500
0250
102 103 104 105 102 103 104 105

CD4 FITC CD4 FITC
Figure 4. Purity of human CD4 T cells. Purity

+
before (top) and after (bottom) negative isolation

Figure 2. Dynabeads products are uniform spherical beads with highly defined and consistent product
®
from peripheral blood mononuclear cells using the
characteristics, which helps ensure that you get truly reliable and reproducible results. Dynabeads Untouched Human CD4 T Cells Kit.
® ™
Molecular Probes® | Flow cytometry products 3

Mouse cell isolation
Gentle tube-based isolation of mouse cells directly from whole blood, spleen, lymph node, or thymus for
any downstream assay, including flow cytometry (Figure 5). Dynabeads products are available for isolating
®
mouse T cells, B cells, NK cells, and dendritic cells.
Isolation options include:
• Positive isolation and cell release

• Negative isolation of untouched cells
• Depletion of unwanted cell types or positive cell isolation for molecular applications
Sample preparation
If you can’t find a ready-to-use product for mouse cell isolation, we have a range of Dynabeads products ®
that can be combined with an antibody of your choice to create a tailored cell isolation tool: streptavidin
Dynabeads , secondary antibody–coated Dynabeads , and surface activated Dynabeads . Learn more at
® ® ®
lifetechnologies.com/mousecellisolation
Find more information online

If you go to lifetechnologies.com/cellisolation, you can find information about our entire line of cell isolation
products, and you’ll also have access to:
• Selection guides for choosing the correct cell isolation product

• Protocols for sample preparation and strategies for cell isolation
• Data showing the performance of our cell isolation products versus other commercially available products
• Help in choosing the correct magnets for your tubes or plates
• Links to videos, brochures, and application notes and to references that cite the use of Dynabeads products ®
Dynabeads® FlowComp™ Mouse CD4 Kit
600 Purity: 105

97.2%
500
Propidium iodide
104
400
Count
300
103
200
100 102 Viability:

86%
0
102 103 104 105 102 103 104 105
CD4+ CD4+
Column-based isolation
400 Purity: 105

350 78.5%
Propidium iodide
300
104
250
Count
200
103
150
100
102 Viability:
50 63%
0
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 Dynabeads FlowComp Mouse CD4 kit (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).

Sample preparation product list
Section Product name Species Target Regulatory Size Cat. No.
cell type status*
Cell preservation 2 mL FIX2
TransFix Reagent
®
NA NA RUO 20 mL FIX20
100 mL FIX100
Red blood cell lysis 25 mL GAS010
Cal-Lyse Whole Blood Lysing Solution
™
NA NA IVD
100 mL GAS010S100
High-Yield Lyse NA NA IVD 500 mL HYL250
Fixation and FIX & PERM Cell Permeabilization Kit
®
NA NA GPR 50 assays GAS003
permeabilization
Sample preparation
200 assays GAS004
Fixation Medium (Medium A) NA NA GPR 100 mL GAS001S100
Permeabilization Medium (Medium B) NA NA GPR 100 mL GAS002S100
Dynabeads cell
®
Dynabeads FlowComp Human CD4 Kit
® ™
Human T cells RUO 3 mL 11361D
isolation
Dynabeads CD4 Positive Isolation Kit
®
Dynabeads Untouched Human CD4 T Cells Kit
® ™
Human T cells RUO 1 kit 11352D
Dynabeads FlowComp Human CD8 Kit
® ™
Dynabeads CD8 Positive Isolation Kit
®
Dynabeads Untouched Human CD8 T Cells Kit
® ™
Human T cells RUO 1 kit 11348D
Dynabeads FlowComp Mouse CD4 Kit
® ™
Mouse T cells RUO 3 mL 11461D
DETACHaBEAD Mouse CD4 Kit
®
Mouse T cells RUO 5 mL 12406D
Dynabeads Untouched Mouse CD4 Cells Kit
® ™
Mouse T cells RUO 2 x 10 mL 11415D
Dynabeads FlowComp Mouse CD8 Kit
® ™
Mouse T cells RUO 1 kit 11462D
Dynabeads Untouched Mouse CD8 Cells Kit
® ™
Mouse T cells RUO 2 x 10 mL 11417D
N/A, not applicable.
* GPR, general-purpose reagent; IVD, in vitro diagnostic use; RUO. For Research Use Only. Not for use in diagnostic procedures.

Instrument set-up and calibration
• Helps to ensure the reliability of optimal daily instrument Flow Cytometry Sub-micron Particle Size
performance
Reference Kit
• Leads to minimal variation for consistent data acquisition
The Flow Cytometry Sub-micron Particle Size Reference
• Compatible with any instrument Kit (Cat. No. F13839) provides a set of green-fluorescent
microsphere suspensions to serve as reliable size references
Flow cytometers are designed to perform quantitative for flow cytometry users. The kit contains six suspensions
measurements on individual cells and other particles with high of polystyrene microspheres, each with a known diameter
precision, speed, and accuracy. As with all high-performance as determined by transmission electron microscopy. The
instrumentation, flow cytometers must be calibrated frequently excitation and emission profile of all the beads is similar
to ensure accuracy and reliability. The stability, uniformity, and to Alexa Fluor 488- or FITC-stained cells (excitation and
®
reproducibility of Molecular Probes microsphere products make

® emission maxima are 505 nm and 515 nm, respectively).
them excellent tools for flow cytometer instrument set-up and
calibration. Learn more about all of these Molecular Probes ®
products at lifetechnologies.com/flow-standards
Number of particles
Alignment
AlignFlow Cytometry Alignment Beads

™
AlignFlow Flow Cytometry Alignment Beads are reliable

™
references for aligning, focusing, and calibrating flow

cytometers. These fluorescently stained polystyrene
microspheres are highly uniform with respect to size and 102 103 104 102 103 104 102 103 104
fluorescence intensity (Figure 6), and are designed to Green Orange Red
approximately replicate the size, emission wavelength, and
Fluorescence intensity
intensity of biological samples. Because the dyes are contained
inside the microsphere’s matrix, instead of on the bead’s Figure 6. AlignFlow beads excited at 488 nm by an argon-ion laser and
™
surface, AlignFlow beads have excellent photochemical

™
monitored in three emission channels. Broad fluorescence emission is
and physical stability, providing reliable reference signals for detected in all three channels. Note the exceptionally small variation in
instrument set-up. The fluorescent dyes have been carefully fluorescence intensity of the beads. Data contributed by Carleton Stewart,
Rosewell Park Cancer Institute.
selected for optimal excitation by laser sources commonly used
in flow cytometry. The AlignFlow beads are available in three
™
versions: for 350–370 nm excitation with UV lasers (Cat. No.

A16502, A16505), for 488 nm excitation with blue lasers (Cat. No. 1.0 µm 6.0 µm 15 µm
A16500, A16503), and for 633 nm excitation with red lasers 300
Number of particles
2.0 µm
(Cat. No. A16501, A16504). Each version is available in two bead 10 µm
sizes: 2.5 µm diameter and 6.0 µm diameter. 200 4.0 µm
Size calibration 100
Flow Cytometry Size Calibration Kit

0
The Molecular Probes Flow Cytometry Size Calibration Kit
®
0 200 400 600 800 1000
(Cat. No. F13838) provides a set of nonfluorescent microsphere FSC
suspensions to serve as reliable size references for cytometry
Figure 7. Flow Cytometry Size Calibration Kit. Histogram analysis of the
users. The kit contains six suspensions of unstained polystyrene
forward scatter intensity (FSC) log channel values of the six polystyrene
microspheres, each with a known diameter, determined by microsphere samples supplied in the Flow Cytometry Size Calibration Kit
transmission electron microscopy. The size of cells in an (Cat. No. F13838) is shown. FSC measurements were performed on a Becton
experimental sample can be estimated by comparing the Dickinson FACScan flow cytometer using excitation at 488 nm.
®
forward scatter (FSC) signals with those of the reference

microspheres. The microspheres function as reproducible size
markers (Figure 7) and can be intermixed with the experimental
sample or used in parallel runs.

The size (or size range) of bioparticles in an experimental sample
can be estimated by comparing their FSC with those of the
Compensation
reference microspheres (Figure 8). The microspheres in each In a perfect world, the fluorescence emission profile for each
component function as reproducible size markers and can be individual fluorophore would be a very intense, narrow peak, well-
used individually (one size), premixed (two to six sizes), intermixed separated from all other emission peaks. In reality, organic dyes
with the experimental sample, or in parallel runs. and fluorescent proteins have broad emission peaks. An example
of the overlap of two commonly used fluorophores is shown in
This kit can be used to verify instrument performance and to Figure 9, which features the emission profiles of Alexa Fluor 488 ®
establish parameters that are suitable for analyzing sub-micron dye and R-phycoerythrin (R-PE). For proper interpretation of the
particles. For example, the kit can be used to check: data collected, it is important to know that the fluorescent signal
being recorded for Alexa Fluor 488 dye is, in fact, coming from
®
• Resolution limit and dynamic range of particle size Alexa Fluor 488 dye and not from R-PE, which happens to emit
®
measurement some light in the same wavelength range. To accurately record

• Sensitivity of forward and side scatter photomultiplier tubes the fluorescence signal for a given fluorophore, it is important to
correct for the emission signal of all dyes, and this correction is
• Level of instrument baseline noise
called compensation.
• Laser and optical alignment and stability
• Stability of the fluidics system We offer two types of compensation kits (Table 1). One type,
the AbC bead kits, is designed for compensation of dyes in

™
immunophenotyping experiments using fluorescently labeled

Cell sorting set-up antibodies. The second type of compensation kit, the ArC Amine ™
Reactive Compensation Bead Kit, is designed for use with cell

viability assays that use amine reactive dyes, such as the LIVE/
Cell Sorting Set-Up Beads DEAD Fixable Dead Cell Stain Kits (see “Cell viability” on page 20
®
The Molecular Probes Cell Sorting Set-Up Beads (Cat. Nos.

®
for more details on these assays).
C16506, C16507, C16508, C16509) are reliable standards for the
set-up and calibration of flow cytometry sorter instruments. The AbC compensation bead kits ™
beads have a diameter of 6 µm (±10%), and thus approximate

the size, emission wavelength, and intensity of many biological • An alternative to using precious samples for setting flow
samples. Consequently, the beads can be used to check cell cytometry compensation
sorter settings such as drop delay and efficiency (cell loss • Highest reactivity to different subclasses of mouse, rat, and
during sorting). The beads can also be used to calibrate a flow hamster immunoglobulin
cytometer’s laser source, optics, and stream flow without wasting
• Fast and simple bead-based flow cytometry compensation
valuable and sensitive experimental material.
• Removal of inconsistencies due to variations in antigen
expression
106 2 µm
AbC bead kits provide a consistent, accurate, and simple-to-
™
1 µm
Particle fluorescence
using 530/30 nm BP
105
use technique for the setting of flow cytometry compensation
104
when using 1) fluorophore-conjugated hamster, mouse,
0.5 µm
0.2 µm
103 0.1 µm
102
100
101
Relative intensity (%)
100 75
102 103 104 105 106
Side scatter 50
25
Figure 8. Flow Cytometry Sub-micron Particle Size Reference Kit. Signals
from (the kit has six particle sizes; only five were used here) five different-sized
0
particles of the Flow Cytometry Sub-micron Particle Size Reference Kit 300 400 500 600 700 800 900
(Cat. No. F13839) were acquired using 488 nm excitation and a 530/30 nm Wavelength (nm)
bandpass (BP) emission filter on the Attune Acoustic Focusing Cytometer.
®
The diameters of the five different green-fluorescent microspheres are Figure 9. Emission profiles of Alexa Fluor 488 dye (green curve) and R-PE
®
identified on a plot of particle fluorescence versus side scatter. (orange curve).

Table 1. Compensation kit selection guide.
Product name Assay type Positive bead Negative bead Cat. No.
AbC Total Antibody

™
Immunophenotyping Hamster, mouse, rabbit, and rat antibodies* No binding capacity A10497
Compensation Bead Kit
AbC Anti-Mouse Bead Kit
™
Immunophenotyping Mouse monoclonal antibodies* No binding capacity A10344
AbC Anti-Rat/Hamster Bead Kit
™
Immunophenotyping Rat and hamster monoclonal antibodies* No binding capacity A10389
ArC Amine Reactive
™
Cell viability assay Amine reactive dyes and LIVE/DEAD Fixable ®
No amine reactive A10346
Compensation Bead Kit Dead Cell Stains capacity
*AbC™ capture beads bind all isotypes.
All products in this table are RUO, For Research Use Only. Not for use in diagnostic procedures.
rabbit, or rat antibodies (AbC Total Antibody Compensation ArC Amine Reactive Compensation Bead Kit
™
™
Bead Kit, Cat. No. A10497; Figure 10); 2) using fluorophore-

• Eliminates the hassle of heat-treating cells
conjugated mouse antibodies (AbC Anti-Mouse Bead Kit, Cat. ™
No. A10344, Figure 11); or 3) using fluorophore-conjugated • Optimized for all LIVE/DEAD Fixable Dead Cell Stain kits ®
rat or hamster antibodies (AbC Anti-Rat/Hamster Bead Kit, ™
• Fast and simple bead-based flow cytometry compensation

Cat. No. A10389). All three kits contain two types of specially
modified polystyrene microspheres: 1) AbC capture beads ™ • An alternative to using precious samples for setting
(also called positive beads), which bind all isotypes of the compensation
specific immunoglobulin, and 2) negative beads, which have no • Accurate and consistent results
antibody binding capacity. After incubation with a fluorophore-
conjugated primary antibody (hamster, mouse, rat, or rabbit,
The ArC Amine Reactive Compensation Bead Kit (Cat. No.
™
depending on the kit used), the two components provide

A10346) provides a consistent, accurate, and simple-to-use
distinct positive and negative populations of beads that can be
technique for the setting of flow cytometry compensation when
used to set compensation (Figure 11).
using any of the LIVE/DEAD Fixable Dead Cell Stain Kits or ®
when using any amine reactive dye. LIVE/DEAD Fixable Dead ®
You can perform compensation with the same fluorophore

Cell Stain Kits (and amine reactive dyes) can be used to evaluate
labeled antibody used for cell staining. The consistent nature of
mammalian cell viability based on the fact that the dye reacts
bead scatter and high surface antibody-binding capacity allows
with cellular amines. The reactive dye can enter the cell via
you to more consistently and accurately set compensation for
compromised membranes of necrotic cells and react with free
any combination of fluorophore-labeled primary antibodies.
amines IgG1in the interior and on the1,000
surface
1,000
ofIgG1the cell, resulting in
Both types of microspheres (the AbC capture beads and ™ 900 900 MouseMouse IgG1
900 900
Rat IgG1
Rat
800
intense
800 MouseMouse
fluorescent staining. In contrast,
IgG2aIgG2a
only
Rat IgG2a
the cell-surface
Rat IgG2a
negative beads) have a diameter of approximately 6 µm (actual

800 800
MouseMouse
IgG2bIgG2b Rat IgG2b
Rat IgG2b
Number of events
Number of events
Number of events
Number of events
amines IgG3of viable cells are available to react with the dye,
700 700 700 700 Rat IgG2c
MouseMouse IgG3 Rat IgG2c
size for each lot is listed on the component vial). The bead 600 600
MouseMouse
IgGM IgGM 600 600 Rat IgGM
Rat IgGM
500 resulting
500 in relatively dim staining.500The500 difference in fluorescence
suspensions are supplied in dropper vials for convenient
intensity between the live and dead cell populations is typically
400 400 400 400
sample application. 300 300 300 300

200 greater
200 than 50-fold. 200 200
100 100 100 100
0 0 0 0
101 101 102 102 103 103 104 104 105 105 106 106 101 101 102 102 103 103 104 104 105 105 106 106
A B C PE fluorescence
PE fluorescence D PE fluorescence
PE fluorescence
1,000 1,000 700 700 800 800
900 900 MouseMouse
IgG1 IgG1 Rat IgG1
Rat IgG1 Armenian hamster
Armenian IgG IgG
hamster RabbitRabbit monoclonal
monoclonal IgG IgG
900 900
800 800 MouseMouse
IgG2a IgG2a Rat IgG2a
Rat IgG2a 600 600 SyrianSyrian
hamster IgG IgG
hamster 700 700 RabbitRabbit polyclonal
polyclonal IgG IgG
800 800
MouseMouse
IgG2b IgG2b Rat IgG2b
Rat IgG2b
Number of events
Number of events
Number of events
Number of events
Number of events
Number of events
Number of events
Number of events
700 700 700 700 Rat IgG2c 500 500 600 600
MouseMouse
IgG3 IgG3 Rat IgG2c
600 600 600 600 Rat IgGM 500 500
MouseMouse
IgGM IgGM Rat IgGM 400 400
500 500 500 500 400 400
400 400 400 400 300 300
300 300
300 300 300 300 200 200
200 200 200 200
200 200
100 100 100 100
100 100 100 100
0 0 0 0 0 0 0 0
101 101 102 102 103 103 104 104 105 105 106 106 101 101 102 102 103 103 104 104 105 105 106 106 101 101 102 102 103 103 104 104 105 105 106 106 101 101 102 102 103 103 104 104 105 105 106 106
PE fluorescence
PE fluorescence PE fluorescence
PE fluorescence
700 700 800 800
Figure
600
10.Armenian
Armenian
Histograms
hamster
600 Syrian Syrian
hamster
IgG IgG
showing the staining of theRabbit
AbC Total
Rabbit
monoclonal Antibody
monoclonal
IgG IgG Compensation Bead Kit. Signal separation of the positive capture beads for mouse (A),
™
hamsterhamster
IgG IgG 700 700 RabbitRabbit
polyclonal
polyclonal
IgG IgG
rat (B), and hamster (C) monoclonal antibodies, and rabbit (D) monoclonal and polyclonal antibodies. Beads were labeled with an optimized amount of each PE
Number of events
Number of events
Number of events
Number of events
500 500 600 600

antibody conjugate and analyzed on an Attune Acoustic Focusing Cytometer using 488 nm excitation and a 574/26 nm bandpass filter.
500 500
®
400 400
400 400
300 300
300 300
200 200
200 200
100 100 100 100
0 0 0 0
101 101 102 102 103 103 104 104 105 105 106 106 101 101 102 102 103 103 104 104 105 105 106 106
PE fluorescence
PE fluorescence

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 distinct
positive and negative populations of beads that can be used to set compensation (Figure 12). The ArC Amine ™
Reactive Compensation Bead Kit can be combined with the AbC Anti-Mouse Bead Kit for use with fluorophore- ™
conjugated mouse antibodies, allowing even more consistent and accurate compensation for multicolor
immunophenotyping experiments that also incorporate a LIVE/DEAD Fixable Dead Cell Stain. ®
Absolute cell counting

Flow cytometry provides a rapid method to quantify cell characteristics. However, most flow cytometers
cannot directly provide the cell concentration or absolute count of cells in a sample. Absolute cell counts have
been widely used in quantifying cell populations and disease progression, including in studies of stem cells.
Absolute cell counts are generally obtained either by combining a separate cell concentration determination
from a hematology analyzer with flow cytometric population data (multiple platform testing) or by adding an
internal microsphere counting standard to the flow cytometric sample (single platform testing). The single
platform method is preferred as it is technically less complicated and it avoids interlaboratory variation and
underestimations, making it more accurate than multiple platform testing.
We offer two products for cell counting—CountBright Absolute Counting Beads and AccuCheck Counting Beads.
™
See Table 2 on the next page to determine which kit is right for your experiment.

A 250 B 250 C
105
CD3-FITC fluorescence
200 200
Events counted
Events counted
104
150 150
103
100 100
50 50 102
0
-102
0
-102 0 102 103 104 105 -350 -102 0 102 103 104 105 -102 0 102 103 104 105
PE fluorescence FITC fluorescence CD56-PE fluorescence
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 B C 350
350
350 300
300
300
Events counted
Events counted
250
Events counted
250
250
200
200
200
150 150
150
100 100
100
50 50 50
0 0 0
-102 0 102 103 104 105 -288 -102 0 102 103 104 105 -715 -102 0 102 103 104 105
LIVE/DEAD® Fixable Violet stain fluorescence LIVE/DEAD® Fixable Green stain fluorescence LIVE/DEAD® Fixable Red stain fluorescence
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.

Table 2. Absolute counting beads selection guide.
Product name Sample Bead size Excitation Emission Parameters Regulatory Cat. No.
type (nm) max (nm) measured status*
CountBright Absolute
™
Any type 7 µm UV to 635 385 to 800 Number of cells RUO C36950
Counting Beads
AccuCheck Counting Whole Bead A: 6.40 µm Bead A: 488 Bead A: 575–585 Number of cells and RUO PCB100
Beads blood Bead B: 6.36 µm Bead B: 635 Bead B: 660–680 accuracy of pipetting
*RUO, research use only. Not for use in diagnostic procedures.
CountBright Absolute Counting Beads

™
AccuCheck Counting Beads
• Compatible with every commercially available instrument • Internal control using ratio of two different color beads
because they are loaded with a wide breadth of indicates pipetting accuracy
fluorochromes • Single platform is preferred over multiple platform testing,
• Easy-to-use protocol that works with multiple cell types leading to consistency in results
including lysed/no-wash whole blood • Easy to validate with most immunophenotyping experiments
• More reliable than multiple platform testing
AccuCheck Counting Beads (Cat. No. PCB100) are an efficient
CountBright Absolute Counting Beads (Cat. No. C36950) are
™
single platform method for absolute cell counting that combines
a calibrated suspension of microspheres that are brightly the advantages of direct flow cytometric immunophenotyping with
fluorescent across a wide range of excitation and emission the use of two different fluorescent beads (A and B beads). These
wavelengths (UV to 635 nm excitation and 385 to 800 nm emission) two fluorospheres are used as a double internal standard for
and contain a known concentration of microspheres. For absolute blood volume calculation. A known volume of AccuCheck Counting
counts, a specific volume of the microsphere suspension is added Beads is added to the same known volume of stained blood in a
to a specific volume of sample, so that the ratio of sample volume lysed/no-wash technique. The beads are counted along with cells.
to microsphere volume is known. The volume of sample analyzed Because the concentration of beads is known, the number of
can be calculated from the number of microsphere events, and cells per microliter (the absolute count) is obtained by relating the
can be used with cell events to determine cell concentration number of cells counted to the total number of fluorescent bead
(Figure 13). In general, at least 1,000 bead events should be events. The cell number is then multiplied by the number of total
acquired to assure a statistically significant determination of fluorospheres per unit of volume. As the AccuCheck Counting
sample volume. Beads system contains two different fluorospheres in a known
proportion, the accuracy of the assay pipetting can be verified
CountBright Absolute Counting Beads can be used with
™
using the proportion of both types of beads.
any sample type, including lysed/no-wash whole blood. The
microspheres in the reagents are approximately 7 µm in diameter
and have settling properties similar to lymphocytes. Sample
preparation steps that can lead to cell or microsphere loss, such
as washes, should be avoided. CountBright beads can be used
™
with either a scatter or fluorescence threshold. When using a

scatter threshold, the microsphere signal should be above the
threshold. The microspheres can be gated by a single parameter,
but a combination of parameters can be used to resolve
microspheres from cells and other events.
Figure 13. CountBright Absolute Counting Beads. A mixture of live and heat-
™
killed Jurkat cells were treated with reagents in the LIVE/DEAD Viability/
®
Cytotoxicity Kit (Cat. No. L3224). CountBright Absolute Counting Beads

™
(Cat. No. C36950) 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.

Instrument set-up and calibration product list.
Section Product name Laser type Ex/Em* Regulatory Size Cat. No.
status †
Alignment AlignFlow Flow Cytometry Alignment

™
A16502
Beads for UV Lasers, 2.5 µm 350–370/
UV RUO 3 mL
AlignFlow Flow Cytometry Alignment
™ 400–470 A16505
Beads for UV Lasers, 6.0 µm
™
A16500
Beads for Blue Lasers, 2.5 µm
Blue 488/515–660 RUO 3 mL
™
A16503
Beads for Blue Lasers, 6.0 µm
™
A16501
Beads for Red Lasers, 2.5 µm
Red 633/645–680 RUO 3 mL
™
A16504
Beads for Red Lasers, 6.0 µm
Size calibration Flow Cytometry Size Calibration Kit NA NA RUO 1 kit F13838
(nonfluorescent microspheres)
Flow Cytometry Sub-micron Particle Blue 505/515 RUO 1 kit F13839
Size Reference Kit
Cell sorting set-up Cell Sorting Set-Up Beads for UV UV 350–375/460 RUO 3 mL C16506
Lasers

Cell Sorting Set-Up Beads for Blue Blue 488/515 RUO 3 mL C16508
Lasers
Cell Sorting Set-Up Beads for Green- Green-yellow 532, 561/575 RUO 3 mL C16509
Yellow Lasers
Cell Sorting Set-Up Beads for Red Red 633/680 RUO 3 mL C16507
Lasers
Compensation AbC Total Antibody Compensation
™
NA NA RUO 1 kit A10497
Bead Kit
AbC Anti-Mouse Bead Kit
™
AbC Anti-Rat/Hamster Bead Kit
™
ArC Amine Reactive Compensation
™
Bead Kit
Absolute cell counting AccuCheck Counting Beads Blue/red 488/575-585 RUO 10 mL PCB100
(Bead A)
CountBright Absolute Counting Beads
™
UV to red UV to 635/385 to 800 RUO 5 mL C36950
N/A, not applicable.
* Excitation and emission maximum wavelengths, in nm.
† RUO, research use only. Not for use in diagnostic procedures.

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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.

Table 3. Fluorophores for flow cytometry.
Fluorophore Laser Emission (nm)
UV Violet Blue Green Yellow Red
405 nm 488 nm 532 nm 561 nm 633/635 nm
Antibody conjugates
Alexa Fluor 350 ®
442
Alexa Fluor 405 ®
421
Pacific Blue™ 455
Pacific Green™ 500
Pacific Orange™ 551
Alexa Fluor 488 ®
519
Fluorescein (FITC) 525
Qdot 605
®
605
Qdot 655
®
655
Qdot 705
®
705
Qdot 800
®
800
Peridinin chlorophyll (PerCP) 678
PerCP-Cy 5.5 ®
695
R-phycoerythrin (R-PE, PE) 575
PE-Texas Red ®
615
PE-Alexa Fluor 610 ®
628
TRI-COLOR (TC, PE-Cy 5)® ®
670
PE-Cy 5.5 ®
694
PE-Alexa Fluor 700 ®
723
PE-Cy 7 ®
767
Allophycocyanin (APC) 660
APC-Cy 5.5 ®
694
APC-Cy 7 767
Antigen detection
®
APC-Alexa Fluor 750 ®

775
Alexa Fluor 647 ®
668
Alexa Fluor 700 ®
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.
MHCII Rat Pacific Blue dye ™
Violet 455 A14901
CD4 Rat Pacific Green dye ™
Violet 500 C11207
B220 Rat Pacific Orange dye ™
Violet 551 RM2630
CD11b Rat FITC Blue 525 RM2801
Foxp3 Rat R-PE Blue 575 N/A
CD3 Hamster PerCP-Cy 5.5 ®
Blue 695 A14784
CD11c Hamster PE-Cy 7 ®
Blue 767 A15849
CD25 (IL-2Rα) Rat APC Red 660 N/A
CD45.2 Mouse APC-Cy 7 ®
Red 767 A18642
CD8 Rat Alexa Fluor 700 ®
Red 719 MCD0829
* All products are Research Use Only. Not for use in diagnostic procedures unless otherwise indicated.
N/A-not available for purchase

Qdot antibody conjugates
®
7 6 58
5,000,000
• Excited by 405 nm or 488 nm, maximizing violet laser use A
Extinction coefficient (cm–1M–1)

1. Qdot® 525
2. Qdot® 565
• Combine with existing organic dyes, increasing the number 4,000,000 3. Qdot® 585
4. Qdot® 605
of detectable parameters 5. Qdot® 625

6. Qdot® 655
3,000,000 4
7. Qdot® 705
• Do not degrade over time like tandem conjugates, allowing 8. Qdot® 800
greater reproducibility 2,000,000 3
• Narrow emission spectra allow for minimal compensation 1,000,000

2
when using a single excitation source 1
0
400 450 500 550 600 650 700 750 800
Wavelength (nm)
Qdot antibody conjugates possess a bright fluorescence
®
emission that makes them well suited for the detection of

low-abundance extracellular proteins. Approximately the same B 1 2 34 5 6 7 8
size as R-PE and compatible with existing organic fluorophore
Fluorescence emission
conjugates, Qdot antibody conjugates can be excited with any
®
wavelength below their emission maximum, but are best excited

by UV or violet light. The narrow, symmetric emission profiles of
Qdot antibody conjugates allow for minimal compensation when
®
using a single excitation source, and their very long Stokes shifts
enable better, more efficient multicolor assays using the 405 nm
violet laser. Available in multiple colors for use in flow cytometry 450 500 550 600 650 700 750 800 850 900
(Figure 15 and Table 5), these advantages make Qdot antibody ®

Wavelength (nm)
conjugates powerful tools for antibody labeling and staining.

Figure 15. Absorption and emission profiles of Qdot labels. (A) ®
Absorption spectra of Qdot labels plotted in terms of molar extinction ®
Learn more about Qdot nanocrystals and their applications in

®
coefficient. (B) Normalized emission spectra of Qdot labels. Numbers ®
flow cytometry at lifetechnologies.com/flow-qdot are the same as those in panel A.
Table 5. Qdot primary antibody conjugates selection guide.

®
Antigen detection
Conjugate ‡
Target* Clone
Qdot 605
®
Qdot 655 ®
Qdot 705
®
Qdot 800
®
CD2 S5.5 Q10172 Q22140 §

Q22141 §
Q22142 §
CD3 S4.1 (AKA 7D6) Q10054 Q10012 Q22149 §
CD4 S3.5 Q10008 Q10007 Q10060

CD4 †
RM4-5 Q10092 Q22163 §
Q22164 §
Q22165 §
CD8 3B5 Q10009 Q10055 Q10059 Q22157 §
CD10 MEM-78 Q10153 Q22135 §

Q22136 §
CD14 TüK4 Q10013 Q10056 Q22137 §

Q10064
CD19 SJ25-C1 Q10306 Q10179 Q22138 §
Q22139 §
CD19 †
6D5 Q22160 §
Q10379 Q22161 §
Q22162 §
CD27 CLB-27/1 Q10065 Q10066

CD38 HIT2 Q10053 Q22150 §
Q22151 §
Q22152 §
CD45 H130 Q10051 Q22154 §

Q10062 Q10156
CD45R (B220) RA3-6B2 Q22166 §
Q10176 Q22167 §
Q22168 §
CD45RA MEM-56 Q10047 Q10069 Q22155 §

Q22156 §
HLADR (class II) Tü36 Q10052 Q22158 §

Q22159 §
Q10063
Isotype control mouse IgG2a Q10014 Q10015 Q10076
* Host for all antibodies is mouse unless otherwise indicated. All products are Research Use Only. Not for use in diagnostic procedures unless otherwise indicated.
† Host is rat.
‡ All product sizes are 100 tests unless otherwise indicated.
§ Product size is 25 tests.

mCherry rat monoclonal antibody conjugates
700
Fluorescent conjugates of our affinity purified rat monoclonal antibodies for mCherry
fluorescent protein (Table 6) can be used to detect native and denatured forms of mCherry 600
or mCherry fusion proteins in flow cytometry applications (Figure 16). Full-length 500
Number of cells
mCherry was used as the immunogen, and the resulting monoclonal IgG2a-isotype
400
antibody detects denatured and native forms of the protein. Because of its improved
brightness, superior photostability, and extremely rapid maturation rate, mCherry 300
monomeric red fluorescent protein is becoming the red fluorescent protein of choice for 200
monitoring physiological processes and detecting transgene expression.
100
0
102 103 104 105 106
Table 6. mCherry rat monoclonal antibody (clone 16D7) conjugates. Pacific Blue fluorescence
™
Dye conjugate Excitation laser color Excitation/Emission (nm) Cat. No. Figure 16. Histogram of gated U2-OS cells
expressing mCherry protein and labeled with
Pacific Blue dye
™
Violet 405/455 M11238 Pacific Blue dye-conjugated rat anti-mCherry
™
Alexa Fluor 488

®
Blue 488/519 M11239 antibody (Cat. No. M11238). Samples were
acquired and analyzed using 405 nm excitation
Alexa Fluor 594 Red 590/617 M11240 and 522/31 nm band pass emission filters on an
Alexa Fluor 647
®
Red 650/668 M11241 Attune Acoustic Focusing Cytometer.
®
Antibody
Antibody labeling
• Polyclonal
• Monoclonal
• Recombinant
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 β-Galactosidase
allow you to use more than one same-species antibody in a single staining experiment. GalT(Y289L)
UDP-GalNAz
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,
Antigen detection
go to lifetechnologies.com/flow-antibodylabeling
N3 N3
N3 N3
The SiteClick antibody labeling system

™
Azide-activated antibody
• Contains reagents to label 100 µg of IgG antibody

• Easy-to-follow step-by-step protocol DIBO alkyne label
• Highly efficient site-specific, reproducible labeling chemistry

• R-PE and Qdot labels for flow cytometry
®
The SiteClick system represents a new paradigm for the universal site-selective labeling
™
of antibodies. This modular, click chemistry–mediated method allows you to enzymatically Site-selectively
label essentially any antibody on its heavy chain N-linked glycans. In contrast to standard labeled antibody
antibody labeling techniques, which can be tedious and inconsistent, the SiteClick site-
™
Figure 17. The SiteClick antibody labeling ™
selective approach produces highly robust and reproducible labeling of antibodies with an system. The first step in the SiteClick antibody ™
impressive choice of detection molecules. labeling process involves removal of terminal

galactose residues from heavy chain N-linked
The site-selective labeling provided by the SiteClick system (Figure 17) prevents
™ glycans using β-galactosidase, exposing
essentially all possible modifiable GlcNAc
disruption of the antigen-binding domain that can occur with traditional amine or thiol residues. Second, the free terminal GlcNAc
reactive labeling reagents and eliminates the need to genetically engineer labeling residues are activated with azide tags by
sites into the antibody prior to modification. This site-selective strategy is especially enzymatic attachment of GalNAz to the terminal
important when labeling monoclonal antibodies that contain lysine residues in or around GlcNAc residues using the GalT(Y289L) enzyme.
the antigen-binding domain, as labeling of these sites can disrupt antigen binding. In the third step, the azide residues are reacted
with the dibenzocyclooctyne (DIBO)-functionalized
Monoclonal antibodies can also have variable sensitivity to disulfide bond–reducing probe of choice (e.g., Alexa Fluor 488 DIBO ®
agents used in reductive cysteine labeling, leaving partially dissociated antibody alkyne). The average degree of labeling is 3–3.5
fragments that result in decreased antibody binding and yield. labels per antibody.

We offer antibody labeling kits with R-PE and an array of Qdot labels (Table 7), which are
®
60
Novice
designed especially for flow cytometry applications. The kits are configured to provide Expert
an easy-to-follow workflow designed to allow novice and experienced scientists alike to Reference R-PE
CD4-positive cells (%)

obtain efficient antibody labeling every time (Figure 18). And, because antibody glycans 40
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 lifetechnologies.com/flow-siteclick
20
Zenon labeling technology

®
Zenon labeling technology provides a versatile, easy-to-use system for labeling mouse
®
0
IgG1, IgG2a, and IgG2b isotypes, as well as rabbit, goat, and human IgG antibodies, with SiteClick™ SiteClick™
our premier Molecular Probes dyes as well as other fluorophores, biotin, photoproteins,
® Qdot® 605 R-PE
and enzymes. This technology offers several advantages over direct chemical labeling, Figure 18. Efficient antibody labeling for both
including: novice and experienced scientists.The SiteClick ™
Qdot 605 Antibody Labeling Kit and SiteClick

® ™
• Speed—the entire labeling procedure takes only 10 minutes R-PE Antibody Labeling Kit were used by both
a novice user and an expert user to label an
• Efficiency—labels nearly 100% of the primary antibodies in solution anti-CD4 monoclonal antibody in triplicate. The
labeled antibodies were used to label CD4-positive
• Economy—labels submicrogram amounts of antibody cells isolated from a single human blood donor,
• Simplicity—no pre- or postlabeling purification of the antibody is required with subsequent analysis by flow cytometry; the
reference sample is a commercially available
• Flexibility—easily use multiple primary antibodies in a single experiment R-PE anti-CD4 antibody conjugate. The data show
percentages of CD4-positive cells relative to
total cells, and the error bars indicate variation
Zenon labeling technology uses a fluorophore-, biotin-, or enzyme-labeled
®
in the triplicate kit labelings for each user. The
Fab fragment directed against the Fc portion of an intact IgG antibody to form a novice user had never before performed protein
noncovalent labeling complex. To ensure their high affinity and selectivity for the bioconjugation, yet obtained labeling efficiencies
Fc portion of the target antibody, the Zenon labeling reagents have been affinity
® equivalent to those obtained by an expert user.
purified during their preparation. Because the Zenon labeling method is based
®
on immunoselectivity, there is no need to remove exogenous proteins or amine

containing buffers from the antibody sample prior to forming the complex. Antibodies
labeled using Zenon technology display fluorescence intensity or enzymatic activity
®
similar to that observed for directly labeled antibody conjugates.

Antigen detection
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 lifetechnologies.com/flow-antibodylabeling
Table 7. SiteClick antibody labeling kits.

™
Product Excitation laser color Emission (nm) Cat. No.

SiteClick R-PE Antibody Labeling Kit
™
Blue 578 S10467
SiteClick Qdot 525 Antibody Labeling Kit
™ ®
UV, violet, blue 525 S10449
™ ®
UV, violet, blue, green 565 S10450
™ ®
UV, violet, blue, green, yellow 585 S10451
™ ®
™ ®
™ ®
™ ®
UV, violet, blue, green, yellow, red 705 S10454
™ ®
UV, violet, blue, green, yellow, red 800 S10455

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 ® †
Alexa Fluor 350 ®

346/442 Z25000 Z25100 Z25200 Z25300 Z25400
Alexa Fluor 405 ®
402/421 Z25013 Z25113 Z25213 Z25313
Alexa Fluor 488 ®
495/519 Z25002 Z25102 Z25202 Z25302 Z25602 Z25402
Alexa Fluor 546 ®
556/573 Z25004 Z25104 Z25304
Alexa Fluor 555 ®
555/565 Z25005 Z25105 Z25205 Z25305 Z25605
Alexa Fluor 568 ®
578/603 Z25006 Z25106 Z25306
Alexa Fluor 594 ®
590/617 Z25007 Z25107 Z25207 Z25307 Z25607 Z25407
Alexa Fluor 647 ®
650/668 Z25008 Z25108 Z25208 Z25308 Z25608 Z25408
Alexa Fluor 680 ®
679/702 Z25010 Z25110 Z25210
Alexa Fluor 700 ®
696/719 Z25011
Classic Dyes †
Pacific Blue ™
410/455 Z25041 Z25156
Pacific Green ™
Z11203
Pacific Orange ™
400/551 Z25256 Z25257
Fluorescein 494/518 Z25042 Z25342
Biotin †
Biotin-XX NA Z25052 Z25152 Z25252 Z25352 Z25452

Phycobiliproteins and Tandem Dyes ‡
R-PE 496 /578

§
Z25055 Z25155 Z25255 Z25355 Z25455
R-PE-Alexa Fluor 610 ®
496 /630
§
Z25020
R-PE-Alexa Fluor 647 ®
496 /668
§
Z25021
Antigen detection
Allophycocyanin (APC) 650/660 Z25051 Z25151 Z25251 Z25351 Z25451
650/775 Z25031
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 lifetechnologies.com/flow-zenon for more information.

Secondary detection
Our extensive selection of secondary detection reagents includes antibodies and streptavidin labeled with our
superior Alexa Fluor dyes, phycobiliproteins, Alexa Fluor dye–phycobiliprotein tandem fluorophores (Figure
® ®
19), Qdot 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.
Find a small selection of conjugates in Table 9. Use our online Secondary Antibody Selection Tool to find the
right secondary detection reagent. With this tool, you can specify target IgG class (and form), host, species
reactivity, and conjugate type to narrow your results. Find your secondary antibody now at
lifetechnologies.com/flow-secondarydetection
Table 9. Secondary detection reagent selection guide.

Dye or label
Dye
Alexa Fluor ®
Pacific Pacific Pacific Alexa Fluor®
PE Alexa Fluor®
Alexa Fluor®
APC Alexa Fluor ®
405 Blue ™
Green ™
Orange ™
488 610–R-PE 647–R-PE 750–APC
Anti-mouse IgG A31553 P10993 P11204 A11001 P852 A20980 A20990 A865 A21006
Anti-rabbit IgG A31556 P10994 P31584 A11008 P2771MP A20981 A20991 A10931
Anti-rat IgG A11006 A10545 A10540
Streptavidin S32351 S11222 S11200 S32365 S11223 S866 S20982 S20992 S868 S21008
S32362*
* Premium grade.
Antigen detection
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 confidential, and we guarantee 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—(RUO) antibody cocktails
For more information, please email Custom Services at custom.services@lifetech.com
Antigen detection

Cell analysis
An extensive array of Molecular Probes stains and kits have been developed to assess
® A
125
cell function, health, and viability. Whether the health of cells is your primary question or Live
Dead
simply a critical factor in getting the right answers to other questions, we have a solution 100
Number of cells
for you. For more information and more products, go to
lifetechnologies.com/flow-cellhealth 75
50
Cell viability 25
Cell viability assays can be used to simply distinguish between live and dead cell 0
populations, to correlate with other cell functions or treatments, or to exclude dead cell 102 103 104 105
populations from analyses. Below we describe two different cell viability assay types LIVE/DEAD®
that use only one channel of the flow cytometer, making them especially useful with B
multicolor flow cytometry (Table 10). For more information and more products go to 150 Dead
lifetechnologies.com/flow-cellviability
Live
Number of cells
LIVE/DEAD Fixable Dead Cell Stain Kits
® 100
• Staining retained after fixation for simple live/dead analysis with intracellular
phenotyping 50
• Fits into almost any staining and phenotyping protocol

• Seven colors to choose from for UV, 405, 488, or 633 lasers 0
102 103 104 105
LIVE/DEAD®
The LIVE/DEAD Fixable Dead Cell Stain Kits (see the product list, page 30) covalently
®
Figure 20. Retention of LIVE/DEAD® Fixable
bind available amino acids but are excluded from the cytosol of live, healthy cells. The Dead Cell Stains after fixation. The LIVE/ DEAD®
dyes react with surface proteins of both live and dead cells, but label proteins throughout Fixable Aqua Dead Cell Stain Kit (Cat. No. L34957)
was used to differentially stain a mixture of live
the cytoplasm of cells with compromised membranes, causing dead cells to fluoresce at
(left peak) and heat-treated Jurkat cells (right
least 50 times brighter than live cells. Because the labeling is covalent, stained cells can peak). Cells in (A) were not fixed; cells in (B) were
be fixed and permeabilized without losing the viability discrimination signal (Figure 20), fixed in 3.7% formaldehyde following staining.
making these reagents ideal if you want to fix and permeabilize samples and maintain Samples were analyzed by flow cytometry using
dead-cell discrimination during subsequent analysis. LIVE/DEAD Fixable Dead Cell
®
405 nm excitation and ~525 nm emission.
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 (A10346; see page 8) for optimal results.
The inadvertent inclusion of sick or dead cells in experiments can dramatically affect
the outcomes. For instance, including dead cells in immunophenotyping analysis
can distort the results, especially for rare phenotypes. Perfetto et al. (2006) showed
Cell analysis
that light-scatter gating during flow cytometry is not enough to exclude all dead cells
from analysis during leukocyte immunophenotyping (see Figure 21 for citation). Using
Molecular Probes LIVE/DEAD Fixable Dead Cell Stains, they were able to efficiently
® ®
exclude dead cells from analysis and, consequently, significantly increase accuracy in
their assays.
Table 10. Cell viability assay selection guide.

Product Target Fixable?* No-wash? Live cell Dead cell Applications
fluorescence fluorescence
LIVE/DEAD Fixable Dead
®
Surface and intracellular Yes No Very dim Very bright Immunophenotyping
Cell Stains proteins
SYTOX Dead Cell stains
®
Nucleic acids No Yes Very dim Very bright Live cell analyses,
dead cell exclusion
* Formaldehyde fixation only.

SYTOX Dead Cell Stains ®
• High-affinity nucleic acid stains for easy dead-cell discrimination

• Multiple colors with minimal spectral overlap for expanded multicolor capabilities
• No wash steps; just add, incubate, and analyze, for simplified protocols
SYTOX Dead Cell Stains (see product list, page 30) are excluded from cells with intact membranes but quickly
®
diffuse into cells that have compromised membranes. Once inside, these dyes bind DNA, which produces a
significant enhancement of their fluorescence; live cells remain nonfluorescent and dead cells fluoresce brightly.
SYTOX stains are available to match widely available excitation sources. These dyes are often used in a “dump
®
channel,” with gating on the viable cells for further analysis (Figure 22). No wash step is required; in fact, SYTOX ®
Dead Cell Stains do not bind covalently to DNA, so dye concentrations must be maintained during analysis.
Lymphocyte gate Viability gate

AA B A
A
200 Live
Live cells Dead cells
FSC-A
FSC-A
Number of cells
150
Dead
100
50
SSC LIVE/DEAD® Fixable Violet fluorescence
CC D
D
CD4–Cy®5.5-PE fluorescence
CD4–Cy®5.5-PE fluorescence
0
0 102 103 104 105
SYTOX® Red fluorescence
B
B
CD3–Alexa Fluor® 488 fluorescence
105
104
CD8–Q705 fluorescence CD8–Q705 fluorescence

103
Figure 21. Exclusion of dead cells eliminates staining artifacts from
analysis. After the application of a lymphocyte gate (A), live and dead cells
were discriminated (B) using LIVE/DEAD Fixable Violet Dead Cell Stain
®
0
Kit (Cat. No. L34955). Note the significant number of dead cells despite a
scatter gate. Subsequent analysis of dead cells (C) and live cells (D) shows 0 103 104 105
the dramatic difference in apparent phenotypes between the two cell
CD8–R-PE fluorescence
populations. Reprinted from Perfetto SP, Chattopadhyay PK, Lamoreaux L,
et al. (2006) J Immunol Methods 313:199–208, with permission from Elsevier. Figure 22. Viable cell gating with SYTOX Dead Cell Stains. A mixture of heat- ®
treated and untreated human peripheral blood leukocytes was stained with
Alexa Fluor 488 dye–conjugated anti–human CD3 antibody, R-PE–conjugated
®
anti–human CD8 antibody, and 5 nM SYTOX Red Dead Cell Stain (Cat. No.
Cell analysis
®
S34859) before analysis by flow cytometry using 488 nm and 635 nm excitation.
(A) Histogram showing distribution of two cell populations, dead cells that
exhibit significant SYTOX Red fluorescence signal, and live cells, which do not.
®
(B) Dual parameter plot of CD8 and CD3 staining after gating on live cells.

Cell proliferation
Cell proliferation and the characterization of agents that either promote or retard cell proliferation are
extremely important areas of cell biology and drug discovery research. We offer both traditional reagents for
assessing cell proliferation (CellTrace Cell Proliferation Kits), as well as the latest technology for measuring
™
new DNA synthesis (Click-iT Plus EdU labeling) (Table 11). For more information and more products, go to
®
lifetechnologies.com/flow-cellproliferation
Click-iT Plus EdU Flow Cytometry Assay Kits

®
• Superior accuracy compared to BrdU assays, with minimal variation (low CV values)
• Streamlined five-step protocol
• Multiplexable with GFP, mCherry, APC, PerCP, PE, and other fluorophores
The growth of cells within a population can be indirectly observed by measuring modified nucleoside
incorporation into newly synthesized DNA. EdU (5-ethynyl-2´-deoxyuridine) is a nucleoside analog that is
incorporated into DNA during synthesis. The Click-iT Plus EdU Flow Cytometry Assay Kits allow detection of ®
EdU (which contains an alkyne) by a copper-catalyzed reaction that produces a stable covalent bond between the
alkyne and a fluorescent dye–labeled picolyl azide. The small size of the picolyl azide detection reagents means
that the fluorescent label has efficient access to the intact DNA without the need for harsh cell treatment.
In the past, DNA synthesis was measured by incorporating the nucleoside analog bromodeoxyuridine (BrdU)
into DNA, followed by detection with an anti-BrdU antibody. Although useful in its time, that method requires
DNA denaturation (using acid, heat, or DNase) to expose the BrdU to the antibody—a step that can adversely
affect sample quality. The Click-iT Plus EdU Flow Cytometry Assay eliminates the need to denature DNA, thus
®
simplifying the assay considerably yet generating comparable results (Figure 23). Click-iT Plus EdU labeling is ®
compatible with fixation protocols.
The Click-iT Plus kits can be used with fluorescent proteins, because the Click-iT Plus reaction uses a modified
® ®
azide in place of the azide used in the original Click-iT reaction. As a result of the modification, the concentration ®
of free copper in the sample is significantly lower and fluorescence signals from fluorescent proteins (e.g., R-PE,
R-PE tandem dyes, and GFP) are not quenched. The speed and accuracy of the Click-iT Plus EdU reaction is ®
comparable to that of the original Click-iT EdU reaction. See the product list on page 30 for available kits.
®
A 200 200
200
B 900 900
900 C
Alexa Fluor® 488 fluorescence
fluorescence
Alexa Fluor® 488 fluorescence
800 800
800 106 6 6
1010
700 700
700
Click-iT® EdU plus
Click-iT®® EdU plus

plus
150 150
150
Number of cells
Number of cells
Number of cells
Number of cells
Number of cells
Number of cells
600 600
600 105 5 5
1010
EdU
500 500
500
Fluor® 488
100 100
100
400 400
400 104 4 4
1010
Alexa Click-iT
300 300
300
Cell analysis
50 5050 200 200

200 103 3 3
1010
100 100
100
0 0 0 0 0 0 102 1010
2 2
101 10101 12
10 2 23
1010
10 3 34
1010
10 4 45
1010
10 5 56
1010
10 6 6
1010 0.0 0.00.0 0.5 0.50.5 1.0 1.01.0 1.5 1.51.5 2.0 2.02.0 0.0 0.00.0 0.5 0.50.5 1.0 1.01.0 1.5 1.51.5 2.0 2.02.0
Click-iT
Click-iT
Click-iT
®
EdU® EdU
plus
®
EdUAlexa
plus
plusAlexa
Fluor
AlexaFluor
®
Fluor
488® fluorescence
488
®
488fluorescence
fluorescence FxCycle
FxCycle
FxCycle
™
Violet
™™
Violet
fluorescence
Violetfluorescence
fluorescence
(x106(x10
) (x10) )
6 6
FxCycle
FxCycle
FxCycle
™
Violet
™™
Violet
fluorescence
Violetfluorescence
fluorescence
(x106(x10
) (x10) )
6 6
Figure 23. Cell proliferation analysis using the Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit and FxCycle Violet Stain. Jurkat cells were treated
® ® ™
with 10 µM EdU for one hour and stained with Alexa Fluor 488 picolyl azide, according to the Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit’s
® ® ®
protocol (Cat. No. C10632), followed by staining with FxCycle Violet Stain (Cat. No. F10347). Cells were then analyzed by flow cytometry using either 488 nm
™
excitation (for Click-iT EdU Alexa Fluor 488 dye) or 405 nm excitation (for FxCycle Violet stain). (A) Histogram demonstrating clear separation of cells in S phase
® ® ™
(DNA synthesis, including EdU incorporation) and cells in either G2/M or G0/G1. (B) Histogram showing DNA content distribution, with G0/G1 and G2/M phase
peaks separated by the S phase distribution using FxCycle Violet stain. (C) Dual parameter Click-iT Plus EdU and FxCycle plot shows co-positive cells that
™ ® ™
provide a direct measurement of the percentage of cells in S phase.

Table 11. Cell proliferation assay selection guide.
Product Target Fixable? Multiplexing? Application
Click-iT Plus EdU Flow Cytometry Assay Kits

®
Incorporation into newly Yes Yes Cell proliferation
synthesized DNA
CellTrace Cell Proliferation Kits

™
Lysine-containing proteins Yes Yes Generational analysis
CellTrace Cell Proliferation Kits

™
• Bright single-peak staining enables visualization of multiple generations

• Well retained in cells for several days post-staining
• No known cytotoxic effect on proliferative ability or biology of cells
• Multiple colors available to easily combine with antibodies or markers of cell function, such as GFP
• Simple, robust staining protocol
The CellTrace family of dyes comprises CellTrace CFSE dye (Cat. No. C34554), CellTrace Violet dye (Cat. No.
™ ™ ™
C34557), and CellTrace Far Red dye (Cat. No. C34564), all of which spontaneously and irreversibly couple to
™
cellular proteins by reaction with lysine side chains and other available amines. When cells divide, the CellTrace ™
dye labeling is distributed equally between the daughter cells, and each successive generation in a population of
proliferating cells is marked by a halving of cellular fluorescence intensity. Eight to ten successive generations
have been identified with both CellTrace CFSE and CellTrace Violet dye (Figure 24). All of the CellTrace dyes
™ ™ ™
can be used in combination with other cell function probes or markers. Because CellTrace Violet and CellTrace ™ ™
Far Red use lasers and detection channels different from those used for green fluorescence, multiplexing with
GFP, fluorescein, or other green-fluorescent probes is possible.
A B C
100 100
4000
80 80
3000
Cell count
Cell count
Cell count
60 60
2000
40 40
1000 20 20
0 0 0
102 103 104 105 106 103 104 105 106 103 104 105 106
CellTrace™ Violet fluorescence CellTrace™ CFSE fluorescence CellTrace™ Far Red fluorescence
Cell analysis
Figure 24. Generational tracing using CellTrace reagents. (A) Cell proliferation was followed for 8 generations using the CellTrace Violet reagent. Human
™ ™
peripheral blood mononuclear cells were harvested and stained with CellTrace Violet reagent prior to stimulation with mouse anti-human CD3 (Cat. No.
™
MHCD0300) and interleukin-2 (Cat. No. PHC0027) for 7 days. The discrete peaks in this histogram represent successive generations of live, CD4 positive cells. The
unstimulated parent generation is indicated in red. (B) and (C) Cell proliferation was followed for 7 generations using the CellTrace CFSE (B) and CellTrace Far ™ ™
Red (C) reagents. Human T lymphocytes were harvested and stained with reagent prior to stimulation with anti-human CD3 (Cat. No. MHCD0300) for 5 days. The
discrete peaks in these histograms represent successive generations of live, SYTOX Green (Cat. No. S34860) negative cells. The unstimulated parent generation
®
is indicated in blue (CellTrace CFSE) or purple (CellTrace Far Red). All analyses were performed using an Attune Acoustic Focusing Cytometer with 405 nm
™ ™ ®
excitation and a 450/40 nm bandpass emission filter for CellTrace Violet detection; with 488 nm excitation and a 530/30 nm bandpass emission filter for CellTrace
™ ™
CFSE detection; and with 638 nm excitation and a 660/20 nm bandpass emission filter for CellTrace Far Red detection. ™

Cell cycle
Detection of DNA content provides a snapshot of cells in a population that are in different A B
105
stages of the cell cycle. Flow cytometry, in conjunction with modeling algorithms, provides 2,000
SYTOX® Blue fluorescence

a powerful tool to assess cells in G0/G1 phase versus S phase, G2/M phase, or showing
Number of cells
104 1,500
polyploidy. Molecular Probes fluorescent dyes allow accurate cell cycle analysis in either
®
Dead
live or fixed cell populations (Table 12). For more information and to see more products, 1,000
go to lifetechnologies.com/flow-cellcycle 103
500
102
Vybrant DyeCycle Stains
® ™
Live
0
0 50 100 150 200 250 0
• Accurate cell cycle analysis in living cells Vybrant DyeCycle Green fluorescence
® ™
Vyb
• Low cytotoxicity for cell sorting and additional live cell
A
experiments B
105
2,000

• Multiple color options for easier multiplexing
Number of cells
• Ability to sort based on phase of cell cycle 104
Dead
1,500
• Ability to identify stem cell side populations using the violet laser 1,000
103
500
Vybrant DyeCycle stains offer the ability to stain for DNA profiling
® ™
10 in live cells, 2
with options for 405, 488, 532, or 633 nm excitation. The dyes are generally used Live
in 0
0 50 100 150 200 250
combination with a dead cell stain (Figure 25) to exclude dead cells from the analysis, but
0 50 100 150 200 250
Vybrant DyeCycle Green fluorescence ® ™

Vybrant® DyeCycle™ Green fluorescence
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. Figure 25. Viable-cell gating with Vybrant DyeCycle ® ™
See the product list on page 30 for the available reagents. stains. Jurkat cells from an overgrown culture
were stained with Vybrant DyeCycle Green Stain ® ™
(Cat. No. V35004) and then SYTOX Blue Dead ®
FxCycle Stains
™
Cell Stain (Cat. No. S34857) and analyzed by flow
cytometry using 488 nm and 405 nm excitation. The
• Allows increased flexibility for multicolor cell cycle studies
histogram (B) was gated on live cells (A) and shows
• Requires little or no compensation with 488 nm excitable dyes DNA content distribution in live cells: G0/G1 and
G2/M phase peaks are separated by the S phase
• Results in tight CV values for more accurate analysis distribution. Inclusion of the dead cells would have
produced aberrant results.
Cell cycle analysis with FxCycle Violet Stain (Cat. No. F10347), FxCycle PI/RNase
™ ™
Staining Solution (Cat. No. F10797), or FxCycle Far Red Stain (Cat. No. F10348) allows
™
easy multiplexing with other cellular assays, such as those measuring cell proliferation
(Figure 23, page 22), immunophenotype, apoptosis, or viability, by freeing up other
lasers and detection channels. The FxCycle stains, intended for cells that are fixed and
™
permeabilized, provide fluorescence signals proportional to the DNA content of each

cell in a population.
Table 12. Cell cycle assay selection guide.

Cell analysis
Product Name Target Live cells? Multiplexing? Application

Vybrant DyeCycle Stains
® ™
DNA Yes Yes Live cell analysis
FxCycle Stains
™
DNA No Yes Fixed cell analysis

Apoptosis
Apoptosis is distinct from necrosis in both the biochemical
and the morphological changes that occur. In contrast to A Control
B Induced
106 106
N N
necrotic cells, apoptotic cells are characterized morphologically 105 105
SYTOX® AADvanced™
by compaction of the nuclear chromatin, shrinkage of the
cytoplasm, and production of membrane-bound apoptotic 104 104
bodies. Biochemically, apoptosis is distinguished by

fragmentation of the genome and cleavage or degradation 102 102
of several cellular proteins. As with cell viability, no single –103 –103
parameter fully defines cell death in all systems; therefore, it is V A V

often advantageous to use several different approaches when –104
–10 4
studying apoptosis. Below we describe several methods and –103 –102 103 105 105 –103 –102 103 105
Molecular Probes products for assessing apoptosis (Table 13). CellEvent Caspase-3/7 Green fluorescence CellEvent® Caspase-3/7 Green fl
®
®
For more information and more products A for apoptosis, go to B

10 Control 106 Induced
lifetechnologies.com/flow-apoptosis
6
N N
105
105
CellEvent Caspase-3/7 Green
® 104 104
Flow Cytometry Assay Kit

102 102
–103 –103
• Optimized caspase-3/7 substrate for apoptosis analysis
V A V A
• Simple, no-wash protocol helps preserve
–10
delicate 4
–104
apoptotic cells –10 –10 10 3 2 3
105 105 –103 –102 103 105 105
CellEvent® Caspase-3/7 Green fluorescence CellEvent® Caspase-3/7 Green fluorescence

• Compatible with both live-cell fluorescence imaging and
formaldehyde-based fixation methods Figure 26. Detection of caspase activity in Jurkat cells, using the CellEvent ®
Caspase-3/7 Green Flow Cytometry Assay Kit. Jurkat cells (human T cell
leukemia) were treated with (A) DMSO or (B) 10 µM camptothecin for 3 hours
Caspases are a family of enzymes that play key roles in initiating before labeling with the CellEvent Caspase-3/7 Green Flow Cytometry Assay
®
and effecting apoptosis, and activation of caspase enzymes is a Kit (Cat. No. C10427). Stained samples were analyzed on the Attune Acoustic ®
distinctive feature of the early stages of apoptosis. We provide Focusing 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 variety of caspase assays for flow cytometry, including the

a 690/50 nm bandpass filter for the SYTOX AADvanced stain (also provided in
® ™
CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (Cat.

®
the kit). Note that treated cells have a higher percentage of apoptotic cells
No. C10427), which detects caspase activity with a substrate that, 7.5 pt than the basal level of apoptosis seen in the control cells (A). A, apoptotic
after being cleaved by caspase-3 or caspase-7, binds to DNA and cells; N, necrotic cells; V, viable cells.
becomes brightly fluorescent (Figure 26).
Table 13. Apoptosis assay selection guide.

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

MitoProbe JC-1 Assay Kit
™
Mitochondrial membrane potential No Yes 2
Violet Ratiometric Membrane Asymmetry Asymmetric membranes No Yes 3
Probe/Dead Cell Apoptosis Kit

Annexin V conjugates and kits
• Conjugated to Molecular Probes dyes for increased sensitivity ® A 104 104
Propidium iodide fluorescence

D
• Conjugates for all available lasers for increased multiplexing capabilities 103 103
• Available as stand-alone reagents or easy-to-use kits

102 102
V V
Another hallmark of apoptosis is the translocation of phosphatidylserine (PS) from 1
A
1
10 10
the cytoplasmic surface of cell membranes, where it normally resides, to the external
surface of cells. Once this translocation has occurred in apoptotic cells, the PS can be
detected by the binding of fluorescently labeled annexin V, a PS-binding protein. We 100
100 101 102 103 104
100
100
offer an array of fluorescent conjugates of annexin V, including conjugates of Molecular Alexa Fluor® 488 fluorescence
Probes bright and photostable Alexa Fluor dyes (Figure 27). Additionally, we have flow
® ®
cytometry–optimized kits that combine some of our other viability and cell function
4
10
B 104

probes with annexin to provide multiparametric data on the apoptoticD state of cells. D
Three of those kits and several annexin V fluorescent conjugates10 are featured in the 3
103
product list on page 31. To find all of the available apoptosis kits, go to
lifetechnologies.com/flow-annexin 10 2
102
V V
A A
MitoProbe JC-1 Assay Kit ™

10 1
10 1
• Easy to use and compatible with existing research protocols

100 100
• Can be used with multiple cell types 100 101 102 103 104 100 101 102 103 104
Alexa Fluor® 488 fluorescence Alexa Fluor® 488 fluorescence
• Available for 488 nm and 633/635 nm excitation
Figure 27. Flow cytometric analysis of Jurkat cells
• Low compensation alternatives using the Alexa Fluor 488 annexin V conjugate and ®
propidium iodide. Jurkat human T cell leukemia

cells were first exposed to 10 µM camptothecin for
The MitoProbe JC-1 Assay Kit (Cat. No. M34152) provides the cationic dye JC-1,
™
four hours (A) or left untreated as control (B). Cells
carbonyl cyanide 3-chlorophenylhydrazone (CCCP, a mitochondrial membrane–potential were then treated with the reagents in the Dead
uncoupler), dimethylsulfoxide (DMSO), and concentrated phosphate-buffered saline (PBS) Cell Apoptosis Kit with Annexin V Alexa Fluor 488 ®
for the study of mitochondrial membrane potential. JC-1 exhibits potential-dependent & Propidium Iodide (Cat. No. V13245) and analyzed
by flow cytometry. Note that the camptothecin-
accumulation in mitochondria, indicated by a fluorescence emission shift from green treated cells have a significantly higher percentage
(~529 nm) to red (~590 nm), due to concentration-dependent formation of red-fluorescent of apoptotic cells (labeled “A”) than the basal level
J-aggregates (Figure 28). Consequently, mitochondrial depolarization is indicated by a of apoptosis seen in the control cells. V, viable
decrease in the red-to-green fluorescence intensity ratio, which is dependent only on cells; D, dead cells.
the membrane potential and not on other factors such as mitochondrial size, shape,
or density, which may influence single-component fluorescence measurements. Use
of fluorescence ratio detection therefore allows researchers to make comparative
measurements of membrane potential and to determine the percentage of cells within
a population that respond to an applied stimulus.
A Untreated B Treated
Cell analysis
- BL-3
JC-1 red fluorescence - BL-3
JC-1 red fluorescence - BL-3

- BL-3
red fluorescence
red fluorescence
red fluorescence
JC-1fluorescence
JC-1JC-1
JC-1 red
JC-1 green
JC-1
JC-1 fluorescence
green
green - BL1-H
fluorescence
fluorescence - BL1-H JC-1
JC-1 JC-1
green green fluorescence
fluorescence
green - BL1-H
fluorescence - BL1-H
Figure 28. Jurkat cells stained with 2 µM JC-1. Cells were stained using reagents in the MitoProbe JC-1 ™
Assay Kit (Cat. No. M34152) for 20 minutes at 37°C and 5% CO2, washed with PBS, and analyzed on the
Attune Acoustic Cytometer using 488 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.

Membrane asymmetry probe
• Accurate apoptotic analysis on trypsinized cells
• Simple 5-minute staining protocol
• Compatible with other blue-excited apoptotic stains
The Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit (Cat. No. A35137) provides
an easy, efficient method for the detection of apoptosis with dead-cell discrimination using a violet laser
flow cytometer (Figure 29). The Violet Ratiometric Membrane Asymmetry Probe detects the membrane
asymmetry changes during apoptosis. It works well on adherent and suspension cells, and correlates with
other indicators of apoptosis, such as caspase detection and changes in mitochondrial membrane potential.
The dye exhibits an excited-state intramolecular proton transfer reaction resulting in dual fluorescence,
with two emission bands corresponding to 530 nm and 585 nm, producing a two-color ratiometric response
to variations in surface charge. The F2N12S probe is combined with SYTOX AADvanced dead cell stain, ® ™
which is capable of passing through the cell membrane only in late apoptotic or necrotic cells, allowing
discrimination from early apoptotic cells.
Unlike annexin-based assays, this assay does not require special buffers or wash steps, and it is less
susceptible to the cell membrane damage commonly found during the physical or chemical removal steps
when assaying adherent cells, therefore providing better data quality.
262,144 262,144
A B
F2N12S green fluorescence
F2N12S green fluorescence
196,608 196,608
A+D A+D
131,072 131,072
65,536 65,536
L L
0 0
0 65,536 131,072 196,608 262,144 0 65,536 131,072 196,608 262,144
F2N12S orange fluorescence F2N12S orange fluorescence

SYTOX® AADvanced™ fluorescence
SYTOX® AADvanced™ fluorescence
105
C D
105
D D
104
10 4
103 L 103
A L
Cell analysis
A
102 102
101 101
0 1,250 2,500 3,750 5,000 0 1,250 2,500 3,750 5,000
F2N12S ratio 585 nm/530 nm F2N12S ratio 585 nm/530 nm
fluorescence x 1,000 fluorescence 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.

Other cell function assays
CellROX Flow Cytometry Kits ®
• Fluorogenic probe that is oxidized to a fluorescent form in the presence of reactive oxygen species (ROS)
• Minimal overlap with fluorophores excited by other laser lines, allowing easy multiplexing
• Cells can be stained in complete media or other appropriate buffer; no need for serum-free media
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.
CellROX reagents are fluorogenic probes for measuring generalized oxidative stress in cells, using conventional
®
fluorescence microscopy, high content screening, microplate fluorometry, or flow cytometry (Figure 30). In a
reduced state, the dyes are nonfluorescent, but upon oxidation, they fluoresce brightly. Molecular Probes kits ®
for flow cytometry include CellROX Green Flow Cytometry Assay Kit (Cat. No. C10492), CellROX Orange Flow
® ®
Cytometry Assay Kit (Cat. No. C10493), and CellROX Deep Red Flow Cytometry Assay Kit (Cat. No. C10491). For ®
more information, go to lifetechnologies.com/flow-cellrox
A B C
105 105

Number of cells
104 104
103 103
102 102
0 0
0 102 103 104 105 0 102 103 104 105 0 102 103 104 105
CellROX® Deep Red fluorescence CellROX® Deep Red fluorescence CellROX® Deep Red fluorescence
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).
Cell analysis

pHrodo E. coli BioParticles Phagocytosis Kits
® ®
• Specifically detect and monitor phagocytosis and endocytosis

• Track antibody internalization with pHrodo conjugates ®
• Track ligand internalization with pHrodo conjugates ®
• Combine with red or green dyes for multiplexed experiments
Proprietary, pH-sensitive Molecular Probes pHrodo dyes are almost nonfluorescent at neutral pH and
® ®
fluoresce brightly in acidic environments, making them ideal for use as pH indicators for a variety of
applications, including studying the processes of phagocytosis and endocytosis. The pHrodo Green E. coli ®
BioParticles Phagocytosis Kit (Cat. No. P35381) and the pHrodo Red E. coli BioParticles Phagocytosis Kit
® ® ®
(Cat. No. A10025; Figure 31) enable the detection of phagocytic activity in whole blood samples and live cell
lines by flow cytometry. The kits include all of the reagents required for assessing particle ingestion and red
blood cell lysis. Additionally, the pHrodo Phagocytosis Particle Labeling Kit (Cat. No. A10026) allows you
®
to label your own sample of bacteria with pHrodo Red dye. To find more information and products, go to
®
lifetechnologies.com/flow-phrodo
120
Number of cells counted
Negative
control
80
Phagocytosed
particles
40
0
100 101 102 103 104
pHrodo dye fluorescence (585 nm)
®
Figure 31. Flow cytometry analysis showing increased fluorescence of granulocytes treated with pHrodo Red BioParticles ® ®
conjugates. A whole blood sample was collected and treated with heparin, and two 100 µL aliquots were prepared. Both aliquots
were treated with pHrodo Red E. coli BioParticles conjugates (used in the pHrodo Red E. coli BioParticles Phagocytosis Kit
® ® ® ®
(Cat. No. A10025)) and vortexed. One sample was placed in a 37°C water bath, and the other sample (negative control) was placed
in an ice bath. After a 15 minute incubation, red blood cells were lysed with an ammonium chloride–based lysis buffer. The samples
were centrifuged for 5 minutes at 500 x g, washed once, and resuspended in Hank's Balanced Salt Solution. The samples were then
analyzed on a BD 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).
Cell analysis

Cell analysis product list
status†
Cell viability LIVE/DEAD Fixable Blue Dead Cell Stain Kit

®
UV 350/450 RUO 200 assays L23105
LIVE/DEAD Fixable Violet Dead Cell Stain Kit
®
Violet 416/451 RUO 200 assays L34955
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit
®
LIVE/DEAD Fixable Yellow Dead Cell Stain Kit
®
LIVE/DEAD Fixable Green Dead Cell Stain Kit
®
Blue 495/520 RUO 200 assays L23101
LIVE/DEAD Fixable Red Dead Cell Stain Kit
®
Blue 595/615 RUO 200 assays L23102
LIVE/DEAD Fixable Far Red Dead Cell Stain
®
Red 650/665 RUO 200 assays L10120
Kit
LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit
®
Red 750/775 RUO 200 assays L10119
LIVE/DEAD Fixable Dead Cell Stain Sampler Kit
®
Various Various RUO 320 assays L34960
SYTOX Blue Dead Cell Stain
®
UV/violet 444/480 ‡
RUO 1 mL S34857
SYTOX Green Dead Cell Stain
®
Blue 504/523 ‡
RUO 1 mL S34860
SYTOX Orange Dead Cell Stain
®
Yellow 547/570 ‡
RUO 1 mL S34861
SYTOX Red Dead Cell Stain
®
Red 640/658 ‡
RUO 1 mL S34859
SYTOX Dead Cell Stain Sampler Kit
®
Various Various
‡
RUO 1 kit S34862
5 x 0.5 mL S10274
SYTOX AADvanced Dead Cell Stain
® ™
Blue 546/647 ‡
RUO
0.2 mL S10349
Cell proliferation Click-iT Plus EdU Pacific Blue Flow
® ™
Violet 404/450 RUO 50 assays C10636
Cytometry Assay Kit
Click-iT Plus EdU Alexa Fluor 488 Flow
® ®
50 assays C10632
Cytometry Assay Kit Blue 495/519 RUO
100 assays C10633
Click-iT Plus EdU Alexa Fluor 647 Flow

® ® 50 assays C10634
Red 650/670 RUO
Cytometry Assay Kit 100 assays C10635
CellTrace Violet Cell Proliferation Kit
™
Violet 405/450 RUO 180 assays C34557
CellTrace CFSE Cell Proliferation Kit
™
Blue 492/517 RUO 1 kit C34554
CellTrace Far Red Cell Proliferation Kit
™
Red 630/661 RUO 1 kit C34564
Cell cycle Vybrant DyeCycle Violet Stain
® ™
Violet 369/437 ‡
RUO 200 µL V35003
Vybrant DyeCycle Green Stain
® ™
Blue 506/534 ‡
RUO 400 µL V35004
Vybrant DyeCycle Orange Stain
® ™
Blue 519/563 ‡
RUO 400 µL V35005
100 assays V10309
Vybrant DyeCycle Ruby Stain
® ™
Red 638/686 ‡
RUO
Cell analysis
400 assays V10273

FxCycle Violet Stain
™
Violet 358/461 RUO 500 assays F10347
FxCycle PI/RNase Staining Solution
™
Blue 535/617 RUO 100 mL F10797
FxCycle Far Red Stain
™
Red 640⁄658 RUO 500 assays F10348
‡ Excitation/emission wavelengths (in nm) when bound to DNA.
§ Emission shift occurs with the concentration-dependent formation of red-fluorescent J-aggregates.

status†
Apoptosis 511/533
CellEvent Caspase-3/7 Green Flow
™
(CellEvent Green) ™ ‡
Blue/green RUO 100 assays C10427

Cytometry Assay Kit 546/647
(SYTOX AADvanced Stain)
® ‡
Annexin V, Pacific Blue conjugate

™
Violet 410/455 RUO 500 µL A35122
Annexin V, FITC conjugate Blue 494/518 RUO 500 µL A13199
Annexin V, Alexa Fluor 488 conjugate
®
Blue 495/519 RUO 500 µL A13201
Annexin V, PE conjugate Blue 496, 546, 565/578 RUO 250 µL A35111
Annexin V, APC conjugate Red 650/660 RUO 250 µL A35110
Annexin V, Alexa Fluor 647 conjugate
®
Red 650/665 RUO 500 µL A23204
410/455
Pacific Blue dye ™
Pacific Blue Annexin V/SYTOX AADvanced

™ ® ™
Violet/blue 546/647 RUO 50 assays A35136

Apoptosis Kit
(SYTOX ®
AADvanced dye) ™
495/519
Dead Cell Apoptosis Kit with Alexa Fluor 488 ® 50 assays V13241
Blue (Alexa Fluor 488 dye)
®
RUO
Annexin V & Propidium Iodide
535/617 (PI) 250 assays V13245
494/519
Dead Cell Apoptosis Kit with FITC annexin V &
Blue (FITC) RUO 50 assays V13242
Propidium Iodide
535/617 (PI)
405⁄530, 585
Violet Ratiometric Asymmetry Probe/Dead (F2N12S)
Violet/blue RUO 100 assays A35137
Cell Apoptosis Kit 546/647 ‡
(SYTOX AADvanced dye)

® ™
MitoProbe JC-1 Assay Kit

™
Blue 514/529, 590 §
RUO 100 assays M34152
Other cell 508/525
function assays (CellROX Green)
®
CellROX Green Flow Cytometry Assay Kit

®
Blue RUO 100 assays C10492
640/658
(SYTOX Red)® ‡
545/565
Green (532 (CellROX Orange)
®
CellROX Orange Flow Cytometry Assay Kit

®
RUO 100 assays C10493
nm) 640/658
(SYTOX Red) ® ‡
640/665
CellROX Deep Red Flow Cytometry
®
(CellROX Deep Red)
®
Red RUO 100 assays C10491

Assay Kit 444/480
Cell analysis
(SYTOX Blue)® ‡
pHrodo Green E. coli BioParticles

® ®
Phagocytosis Kit Blue 509/533 RUO 100 assays P35381
pHrodo Green S. aureus BioParticles

® ®
Blue 509/533 RUO 100 assays P35382

Phagocytosis Kit
pHrodo Red E. coli BioParticles
® ®
Blue 560/585 RUO 100 assays A10025

Phagocytosis Kit
pHrodo Phagocytosis Particle Labeling Kit
®
Blue 560/585 RUO 100 assays A10026

‡ Excitation/emission wavelengths (in nm) when bound to DNA.
§ Emission shift occurs with the concentration-dependent formation of red-fluorescent J-aggregates.

Notes

BOUNDARY-BREAKING
ACOUSTIC FOCUSING CYTOMETRY
Introducing the Attune NxT Acoustic Focusing Cytometer—

®
a high-performance package that’s flexible enough for any lab

The Attune NxT Acoustic Focusing Cytometer combines unmatched precision, speed,
®
and sensitivity, in a modular design that can grow with you. With up to 4 lasers and
14 colors, the Attune NxT cytometer is designed to accommodate most experimental
®
design needs and lab budgets. Boundaries, beware.
See the next big thing in flow cytometry at

lifetechnologies.com/attune
Brand elem
Life logo and brand marque

Life Technologies logo
The Life logo contains three elements:
the Life script, technologies descriptor
and the endorser. The visual relationship
between the logo and the endorser is
fixed and is not to be modified.
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For Research Use Only unless indicated. Not for use in diagnostic procedures. © 2014 Thermo Fisher Scientific Inc. All rights reserved. All
trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Cy is a registered trademark of GE Healthcare.
FACScan and FACSCalibur are trademarks or registered trademarks of Becton, Dickinson and Company. TransFix is a registered trademark of
® ™
Sheffield Teaching Hospitals NHS Trust. FIX & PERM is a registered trademark of Nordic Immunological Laboratories. CO010599 0914
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Molecular Probes flow cytometry

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Molecular Probes® | Flow cytometry products

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guide presents an overview of primary antibody conjugates, cell

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Fixative-Free High-Yield Lyse -103 103 104

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Figure 4. Purity of human CD4 T cells. Purity

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