Kits and Templates

for the BD Accuri™ C6

As Easy as Cell Analysis is Going to Get

 As easy as cell analysis is
going to get
Table of Contents
Cell Biology: Apoptosis 4
Cell Cycle and DNA 6
Immunology: Naïve/Memory T Cells 8
Intracellular Cytokines 10
Tregs 12
Stem Cells: Pluripotent Stem Cells 14
Bead-Based Assays: BD™ Cytometric Bead 16
Array (CBA)
Microbiology: Water Quality 18
Instrument Setup: Validation Beads 19
2
The analytical power and versatility of today’s laser-
based flow cytometry systems have unlocked the
mysteries of cell biology and empowered entirely
new fields of research.
Reagent Kits
BD reagent kits are preconfigured to conduct many
common flow cytometry assays for applications such as
immunophenotyping, apoptosis, cell cycle, stem cells,
microbiology, intracellular cytokines, and more. In addition
to preselected fluorescent antibodies, the kits include
buffer systems, other reagents, and protocols needed for
acquisition and analysis.
Software Templates
Free BD Accuri™ C6 software templates are matched to
each kit. A template is a predefined workspace that includes
markers, regions, gates, labels, parameter names, run
criteria, and compensation settings for an assay using the kit.
These settings provide a shortcut to quick and easy setup
and analysis.
As a result, flow cytometry has become a staple of modern laboratories around the world. Now, BD makes it
even easier to apply the power of flow cytometry to your research with ready-to-go reagent kits, protocols,
and free software templates for the BD Accuri™ C6 personal flow cytometer. 

BD Accuri C6 Using Kits and Templates

The analytical power and versatility of today’s laser-based
flow cytometry systems have unlocked the mysteries of cell
biology and empowered entirely new fields of research.
As a result, flow cytometry has become a staple of modern
laboratories around the world. Innovations in ease of use
reflected in the BD Accuri C6 flow cytometer make these
powerful capabilities accessible and affordable.
The compact footprint and transportable weight of the
BD Accuri C6 also make it a valuable personal use tool for
experienced researchers who want a cytometer to be easily
available when and where they need it.
Ease of use is a hallmark of the system because it is designed
for the non-flow expert. Using the BD Accuri C6, customers
can begin collecting and analyzing data with the help of
a quick start guide. The intuitive interface of the software
guides the user through workflows.
Template Home Page
Reagent kits and their associated software templates are listed
at bdbiosciences.com/go/templates. There you can browse and
order the kits, download the templates, examine sample data,
replay webinars, and review product information sheets.
Using Templates
Once you have downloaded a template, select File > Open
workspace or Template and open it. Gate positions, zoom
level, thresholds, and compensation, run, and fluidics settings
are all fully adjustable and may require optimization for
different sample types. Once the settings are optimized for
your experiment, click Run to begin data acquisition.
Opening a template
Select File > Open workspace or template. All settings are
fully adjustable.

The system takes in data digitally over a 7-decade dynamic

range, allowing researchers to easily re-analyze data after
it is collected. This helps ensure that, should data need to
be re-examined to accommodate new research, the data is
always available.

The BD Accuri C6 flow cytometer is small enough to easily

fit on a benchtop and can be placed in a laminar flow hood.
It measures 11 x 14.75 x 16.5 inches (H x W x D) (27.9 x 37.5 x
41.9 cm) and weighs just 30 pounds (13.6 kg).

3
 Cell Biology: Apoptosis BD apoptosis kits, protocols, and software templates
for the BD Accuri C6 simplify the detection of apoptosis
at different stages. The kits support studies involving
Annexin V, JC-1, and active caspase-3, and include
buffers and other agents needed for acquisition and
analysis.

The BD Pharmingen™ Annexin V FITC and PE Apoptosis
Detection Kits (Cat. Nos. 556570 and 559763) detect
externalization of phosphatidylserine (PS) molecules on the
plasma membrane, one of the first signs of the apoptotic
process.
The BD Pharmingen™ BD™ MitoScreen (JC-1) Kit (Cat. No.
551302) detects apoptosis from changes in mitochondrial
membrane potential (ΔΨm) due to the accumulation of JC-1
aggregates.
The BD Pharmingen™ Caspase-3 PE Assay Kit (Cat. No.
550914) detects apoptosis from the presence of cleaved
(activated) caspase-3, a key apoptotic protease that cleaves
and activates other caspases as well as other cellular targets.
Apoptosis (programmed cell death) is an important
biological process for both development and normal tissue
homeostasis. Dysregulation of apoptotic pathways can lead
to disease.
Flow cytometry is an especially powerful method for
detecting apoptosis because researchers can gain
quantitative data on both apoptotic and dead cells within
whole populations and cell subsets. BD offers multiple
methods for detecting cells at various stages of apoptosis, as
well as a broad portfolio of reagents to identify cell subsets.
For a broader review of apoptosis detection on the
BD Accuri C6, see the BD Biosciences technical bulletin,
Multiple Methods for Detecting Apoptosis on the
BD Accuri™ C6 Flow Cytometer (March 2012), available
online at bdbiosciences.com.

BD Pharmingen Annexin V FITC Apoptosis Detection Kit II Quantity Number of Tests Cat. No.
Purified Recombinant Annexin V 100 μg
FITC Annexin V 0.5 mL
100 tests 556570
Propidium Iodide Staining Solution 2.0 mL
10X Annexin V Binding Buffer 50 mL

BD Pharmingen Annexin V FITC Apoptosis Detection Kit I Quantity Number of Tests Cat. No.
PE Annexin V 0.5 mL
7-AAD 2.0 mL 100 tests 559763
10X Annexin V Binding Buffer 2.0 mL

BD MitoScreen (JC-1) Kit Quantity Number of Tests Cat. No.

JC-1 50 mL
100 tests 551302
10X Assay Buffer 60 mL

BD Pharmingen Caspase-3 PE Apoptosis Kit Quantity Number of Tests Cat. No.

PE Rabbit Anti-Active Caspase-3 20 μL/test
BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution 65 mL 100 tests 550914
BD Perm/Wash™ Buffer (10X Solution) 65 mL

All kits and their associated software templates are available at bdbiosciences.com/go/templates.

4
 Detecting Mitochondrial Membrane Changes Using JC-1 Detecting Apoptosis Using Caspase Activation
A A02 K562 untreated JC-1 B A02 K562 untreated JC-1 C A03 K562 CCCP JC-1 A A05 DMSO B A05 DMSO C A06 Camptothecin
4,000,000 5,292,813

,595,118
Gate: [No Gating] Gate: (P1 in all) Gate: (P1 in all) Gate: [No Gating] Gate: (P1 in all) Gate: (P1 in all)

1,000

1,000
10 7.2

10 7.2
V4-L V4-R V4-L V4-R
97.0% 3.0% 74.2% 25.8%

Cell Biology
P4 P4

10 6

10 6
83.0% 7.7%

1,000,000 2,000,000

500

500
FL2 JC-1-A

FL2 JC-1-A
2,000,000

10 5

10 5

Count

Count
SSC-A
SSC-A

P1 P1
84.8% P5 P5 96.3%
15.0% 91.4%
10 4

10 4
10 3.3

10 3.3
0

0
599,187 5,000,000 10,000,000 14,280,607 10 3.7 10 5 10 6 10 7.2 10 3.7 10 5 10 6 10 7.2 0 2,000,000 4,000,000 6,990,507 10 2.7 10 3 10 4 10 5.2 10 2.7 10 3 10 4 10 5.2
FSC-A FL1 JC-1-A FL1 JC-1-A FSC-A FL2 Active caspase-3 PE-A FL2 Active caspase-3 PE-A

BD MitoScreen (JC-1) Kit (Cat. No. 551302) analysis on the BD BD Pharmingen Caspase-3 Assay Kit (Cat. No. 550914) analysis on
Accuri C6. the BD Accuri C6.
K562 cells (human chronic myelogenous leukemia; ATCC CCL-243) Jurkat cells (human T-cell leukemia; ATCC TIB-152) were treated
were treated with 100 μM of CCCP (in DMSO) for 5 minutes at with 6 μM of camptothecin or 0.1% DMSO (negative control) for
37ºC to induce mitochondrial membranes to decouple. The cells 4 hours to induce apoptosis. Cells were permeabilized, fixed, and
were stained with JC-1 and washed according to the kit protocol, stained according to the kit protocol, acquired on a BD Accuri C6
acquired on a BD Accuri C6 flow cytometer using the kit template, flow cytometer using the kit template, and analyzed using
and analyzed using BD Accuri C6 software. Results: Compared to BD Accuri C6 software. Results: Camptothecin treatment (C)
untreated controls (B), CCCP treatment (C) resulted in a shift in resulted in an increase in active caspase-3 expression compared to
mitochondrial membrane potential (red to green). Because JC-1 the DMSO control (B), which was almost completely negative.
staining patterns can vary for different cell types and experimental
conditions, P4 and P5 gate boundaries should be adjusted
according to the kit guidelines.

Detecting Early-Stage Apoptosis

Using Annexin V
B02 DMSO FITC + PI B02 DMSO FITC + PI
,695,318

Gate: [No Gating] Gate: (P1 in all)

10 7.2

Q5-UL Dead
0.1% 11.3%
10 6
1,000,000

FL3 Propidium Iodide-A

10 5

P1
88.4%
10 4
SSC-A
500,000

10 3
10 2

Live Apoptotic
85.4% 3.2%
10 1
0

0 5,000,000 10,067,283 10 1 10 2 10 3 10 4 10 5 10 6 10 7.2

FSC-A FL1 Annexin V FITC-A

B03 Camptothecin FITC + PI B03 Camptothecin FITC + PI

,695,318

Gate: [No Gating] Gate: (P1 in all)

10 7.2

Q5-UL Dead
0.1% 9.3%
10 6
1,000,000

FL3 Propidium Iodide-A

10 5

P1
53.1%
10 4
SSC-A
500,000

10 3
10 2

Live Apoptotic
62.3% 28.3%
10 1
0

0 5,000,000 10,067,283 10 1 10 2 10 3 10 4 10 5 10 6 10 7.2

FSC-A FL1 Annexin V FITC-A

BD Pharmingen Annexin V FITC Apoptosis

Detection Kit (Cat. No. 556570) analysis on
the BD Accuri C6.
Jurkat cells (human T-cell leukemia; ATCC
TIB-152) were treated with 6 μM of
camptothecin or 0.1% DMSO (negative
control) for 4 hours to induce apoptosis.
Cells were stained with FITC Annexin
V and PI according to the kit staining
protocol, acquired on a BD Accuri C6 flow
cytometer using the kit template, and
analyzed using BD Accuri C6 software.
Results: Camptothecin treatment (lower
plots) resulted in an increase in early
apoptotic cells (PI–Annexin V+, shown in
green) compared to the DMSO control
(upper plots). Dead or late-stage apoptotic
cells (PI+Annexin V+, red) and live cells (PI–
Annexin V–, black) were easily distinguished.
5
 Cell Biology:
Cell Cycle and DNA
The BD Cycletest™ Plus DNA Reagent Kit (Cat. No. 340242)
uses PI and other active agents to obtain precise ploidy
and cell cycle measurements using isolated cell nuclei.
Researchers can use it to estimate the DNA index (DI) and
cell cycle distribution of DNA stemlines and identify those
with abnormal ploidy.
The BD Pharmingen™ FITC and APC BrdU Flow Kits (Cat.
Nos. 559619 and 552598) use 7-AAD and BrdU to provide
high-resolution cell cycle measurements. Researchers
can use them to identify and analyze actively cycling cell
subpopulations, and to examine cell cycle kinetics.
6
BD cell cycle/DNA kits, protocols, and software
templates for the BD Accuri C6 simplify the assessment
of cell cycle and DNA status.
Flow cytometry has become an essential methodology for
assessing cell cycle, cell proliferation, and DNA content.
Multicolor flow cytometric assays allow researchers
to investigate these facets of cell status—along with
other cellular events, such as apoptosis, DNA damage,
protein phosphorylation, or cytokine secretion—within
heterogeneous cell populations.
Dyes such as PI and 7-AAD, which bind to DNA, can trace
changing DNA levels and generate characteristic cellular
DNA content profiles. To determine aneuploidy of abnormal
cells, normal or peripheral blood mononuclear cells (PBMCs)
can be stained simultaneously as a reference.
When cells are incubated in the presence of BrdU, an analog
of the DNA precursor thymidine, the molecule is incorporated
into newly synthesized DNA. BrdU assays can assess cell
proliferation and apoptosis as well as cell cycle distribution.
 The kits include key DNA reagents such as propidium iodide (PI), 7-amino actinomycin D (7-AAD), and
bromodeoxyuridine (BrdU), along with buffers and antibodies needed for acquisition and analysis. The panels
are compatible with other markers that allow researchers to characterize the subpopulations found.

Cell Biology
BD Cycletest Plus DNA Reagent Kit Quantity Number of Tests Cat. No.
Solution A: Trypsin in spermine tetrahydrochloride detergent buffer 10 mL
Solution B: RNase A and trypsin inhibitor in spermine buffer 8 mL
40 tests 340242
Solution C: Propidium iodide (PI) in spermine buffer 8 mL
Buffer Solution: Dimethyl sulfoxide (DMSO) in sucrose-sodium citrate 3 x 50 mL

BD Pharmingen FITC or APC BrdU Flow Kit Quantity Number of Tests Cat. No.
FITC or APC BrdU (1o mg/mL) 5 x 0.5 mL
DNase 5 x 300 μL
Fluorochrome-conjugated anti-BrdU antibody 1 x 65 μL
559619 (FITC)
BD Cytofix/Cytoperm buffer 1 x 25 mL 50 tests
552598 (APC)
BD Perm/Wash buffer (10X) 2 x 25 mL
BD Cytofix/Cytoperm™ Plus permeabilization buffer 1 x 10 mL
7-AAD 1 x 1 mL

Identifying Cell Cycle Phases Using

BrdU and 7-AAD Assessing Cell Cycle and Ploidy Using PI
A A04 PI FITC BrdU + 7-AAD B A05 PI APC BrdU + 7-AAD A A03 K562 B A03 K562 C A05 PBMC + K562
Gate: (P3 in all) Gate: (P3 in all) Gate: (P2 in all) Gate: (Singlets in (P2 in all)) Gate: (Singlets in all)
1,600
10 6.5

500
10 7

10 7

S PBMCs G0/G1
S
400

6.4% MFI=396,087
10 6

6.8% Singlets G0/G1

10 6

93.9% 44.6%
FL2 Propidium Iodide-H

1,000
10 6
FL4 BrdU APC-A
FL1 BrdU FITC-A
10 5

G0/G1 G2/M K562 G0/G1

10 5

70.9% 21.3% MFI=571,768

Count

Count
200

G2/M S
34.5%
500

3.2%
10 4

10 4

G0/G1
G2/M 69.6%
4.0%
10 2.7

10 5.1
10 3

0 50,000 120,000 0 50,000 100,000 10 5.3 10 6 10 6.6 400,000 1,000,000 1,300,000 300,000 500,000 1,000,000 1,300,000
FL3 7-AAD-A FL3 7-AAD-A FL2 Propidium Iodide-A FL2 Propidium Iodide-A FL2 Propidium Iodide-A

BD Pharmingen FITC and APC BrdU Flow BD Cycletest Plus DNA Reagent Kit (Cat. No. 340242) analysis on
Kits (Cat. Nos. 559619 and 552598) the BD Accuri C6.
analysis on the BD Accuri C6. K562 leukemia cells (incorporating the Philadelphia translocation)
Human peripheral blood mononuclear were cultured and stained according to the kit protocol, acquired
cells (PBMCs) were stimulated, expanded, on a BD Accuri C6 flow cytometer using the kit template, and
restimulated, and labeled with 20 μM analyzed using BD Accuri C6 software. A. K562 cells were gated to
of BrdU during the final hour. After exclude aggregates on a PI FL2-A vs PI FL2-H plot. B. A PI histogram
harvesting and staining the cells with of the gated K562 singlets distinguishes cells at the G0/G1, S, and
7-AAD and either FITC or APC anti-BrdU G2+M cycle phases. Gating of cell cycle phases is approximate and
according to the kit protocol, samples can be refined using software analysis. C. Staining and analyzing
were acquired on a BD Accuri C6 flow normal PBMCs along with the K562 cells can quantify their
cytometer using the kit template, and aneuploidy by gating on their G0/G1 peaks. The ratio of the MFIs of
analyzed using BD Accuri C6 software. the two peaks, called the DNA Index (DI), serves as a measure of
Cell cycle phases are clearly distinguished aneuploidy—in this case 1.4.
in plots showing (A) 7-AAD vs BrdU FITC
and (B) 7-AAD vs BrdU APC. Cells in black
(to left of G0/G1 gate) contain less DNA,
which may indicate cell death.

7
 Immunology:
Naïve/Memory T Cells
The BD Multitest™ Human CD45RA/CD45RO/CD3/CD4
Kit (Cat. No. 340571) is a convenient antibody cocktail to
differentiate between naïve and memory T cells using the
classic markers CD45RO and CD45RA. The lyse/no-wash
protocol is fast and easy to use.
The BD Multitest™ Human CD45RA/CD62L/CD3/CD4 Kit
(Cat. No. 340977) also distinguishes naïve from memory CD4
T cells in a convenient antibody cocktail, and adds a CD62L
(L-selectin) antibody to assess activation of memory cells.
The BD Pharmingen™ Human Naïve/Memory T Cell Panel
(Cat. No. 561438) also distinguishes naïve from memory CD4
T cells in a convenient antibody cocktail, and adds a CD197
(CCR7) antibody to identify central and effector memory
cells. It may be combined with CD27, CD28, and other
markers to assess antigen experience, depending on the
level of activation.
8
BD naïve/memory T-cell kits, protocols, and
software templates for the BD Accuri C6 simplify the
identification of human naïve and memory T cells and
their subsets. The kits include antibodies for key T-cell
markers such as CD45RA, CD45RO, CD62L, and CD197,
which can not only distinguish memory from naïve T
cells but subset them into activated vs non-activated or
central vs effector cells.
The immune system’s ability to respond with greater
intensity upon re-exposure to antigens forms the basis
of immunological memory. The understanding of
immunological memory is important for the study of
vaccine development, infectious disease, and immune
reconstitution.
Relative populations of different T-cell subsets, activation
markers, and growth factors can provide a useful
measurement of immune response to antigens. Memory
T cells, derived from naïve T cells upon activation, are
characterized phenotypically in humans by high levels of
CD45RO expression. Central memory cells retain the lymph
node (LN)-homing receptors CD197 (CCR7) and CD62L
(L-selectin) as they develop from the naïve T cells. Effector
memory cells, on the other hand, downregulate CD62L and
express a diverse set of homing receptors, which enables the
cells to pass through non-lymphoid tissues.1
1. Appay V, van Lier RA, Sallusto F, Roederer M. Phenotype and function of human T
lymphocyte subsets: consensus and issues. Cytometry A. 2008;73:975-983.
 BD Multitest Human CD45RA/CD45RO/Cd3/CD4 Kit Clone Quantity Number of Tests Cat. No.
Human CD3 PerCP SK7
Human CD4 APC SK3
20 μL/test 50 tests 340571
Human CD45RA FITC L48
Human CD45RO PE UCHL-1

Immunology
BD Multitest Human CD45RA/CD62L/CD3/CD4 Clone Quantity Number of Tests Cat. No.
Human CD3 PerCP SK7
Human CD4 APC SK3
20 μL/test 50 tests 340977
Human CD45RA FITC L48
Human CD62L PE SK11

BD Pharmingen Human Naïve/Memory T Cell Panel Clone Quantity Number of Tests Cat. No.
Human CD3 APC-H7 (optional drop-in; not used on BD Accuri C6) SK7 5 μL/test
Human CD4 PerCP-Cy™5.5 SK3 5 μL/test
50 tests 561438
Human CD45RA FITC HI100 5 μL/test
A C11
Human CD197 (CCR7) Alexa Fluor® 647 Gate: (CD3+ in all) 150503 5 μL/test
10 5.9

All kits and their associated software templates are available on the BD Biosciences website. Naive
10 5

11.6%
10 4
FL4 CD4 APC-A
10 3

Differentiating Naïve vs Distinguishing Activated vs Distinguishing Central vs

10 1.5 10 2

Memory T Cells 10 1.4 10 2 10 3 10 4

FL1 CD45RA FITC-A
10 510 5.4
Non-Activated Memory Cells Effector Memory Cells
A B
A C11 B C11 A B06 B B06 C06 C06
500,000 1,000,000 1,500,000 1,997,288

Gate: (CD3+ in all) Gate: (CD3+ in all) Gate: (CD3+ in all) Gate: (CD3+CD4+ in (CD3+ in all)) Gate: [No Gating] Gate: (CD4+ in all)

10 4.8
10 5.9
10 5.9

10 6.2

10 5 10 5.5

Non-Activated Memory Naive Central Memory Naive

Memory CD3+CD4+ 52.4%
Memory 25.5% 22.5% 44.6%
Naive 51.7% 72.9% 52.4%
10 5

10 5

11.6%
10 5

10 4
10 4

FL4 CD197 AF 647-A

10 4
10 4

10 4
FL4 CD4 APC-A

FL4 CD4 APC-A

FL4 CD4 APC-A

FL2 CD62L PE-A

SSC-A
10 3
10 3

10 3
10 3

10 3

10 2

CD4+
10 2

6.9%
10 1.5 10 2

10 1.5 10 2

Activated Memory Q1-LR Effector Memory Q1-LR

21.4% 0.7% 31.6% 1.3%
10 1.9
10 1.3
10 1

10 1.4 10 2 10 3 10 4 10 510 5.4 10 1.4 10 2 10 3 10 4 10 510 5.4 10 1 10 2 10 3 10 4 10 5 10 6.2 10 1.3 10 2 10 3 10 4 10 5.2 10 2 10 3 10 4 10 4.7 10 1.5 10 2 10 3 10 4 10 5.1
FL1 CD45RA FITC-A FL2 CD45RO PE-A FL3 CD3 PerCP-A FL1 CD45RA FITC-A FL3 CD4 PerCP Cy5.5-A FL1 CD45RA FITC-A

B C11
Gate: (CD3+ in all)
BD Multitest Human CD45RA/CD45RO/CD3/ BD Multitest Human CD45RA/CD62L/CD3/ BD Pharmingen Human Naïve/Memory T
10 5.9

Memory
CD4 Kit (Cat. 51.7% No. 340571) analysis on the CD4 Kit (Cat. No. 340977) analysis on the Cell Panel (Cat. No. 561438) analysis on
10 5

BD Accuri C6. BD Accuri C6. the BD Accuri C6.

10 4
FL4 CD4 APC-A

Human peripheral blood was stained Human peripheral blood was stained Human peripheral blood was stained
according to the kit procedure, acquired according to the kit procedure, acquired according to the kit procedure, acquired
10 3

on a BD Accuri C6 flow cytometer using on a BD Accuri C6 flow cytometer using the on a BD Accuri C6 flow cytometer using
10 1.5 10 2

the
1.4 2
kit template,
3 4
and analyzed using
5 5.4
kit template, and analyzed using BD Accuri the kit template, and analyzed using
10 10 10 10 10 10

BD Accuri C6 software with a gate set on

FL2 CD45RO PE-A
C6 software with a gate set on CD3+ BD Accuri C6 software. (The optional
CD3+ lymphocytes. Results: The CD3+ T lymphocytes. Results: A. An additional drop-in reagent CD3 APC-H7 was not used.)
cells were characterized as either (A) naïve gate was drawn around the CD3+CD4+ Results: A. A gate was drawn around
(CD4+CD45RA+, blue) or (B) memory cells T-cell population. B. The CD3+CD4+ T the CD4+ T-cell population. B. The CD4+ T
(CD4+CD45RO+, red). cells were characterized as either naïve cells were characterized as either naïve
(CD45RA+CD62L+, blue), non-activated (CD45RA+CD197+, blue), central memory
memory (CD45RA–CD62L+, red), or activated (CD45RA–CD197+, red), or effector memory
memory cells (CD45RA–CD62L–, purple). cells (CD45RA–CD197–, purple).

9
 Immunology: BD T-cell cytokine kits, protocols, and software
templates for the BD Accuri C6 simplify the detection
Intracellular Cytokines of cytokines and cell surface markers. The kits
support studies involving the production IFN-γ, IL-4,
and IL-17A by activated Th1, Th2, Th17, and other
cells. They include antibodies to surface markers that
help characterize the cytokine-producing cells, along
with buffers and transport inhibitors needed for
acquisition and analysis.

The BD Pharmingen™ Human Th1/Th2/Th17 Phenotyping
Kit (Cat. No. 560751) is designed to assess the differentiation
of naïve T cells into Th1, Th2, and Th17 cells, which secrete
IFN-γ, IL-4, and IL-17A, respectively.
The BD FastImmune™ Human IFN-γ/CD69/CD8/CD3 Kit
(Cat. No. 346048) measures the response of activated CD8+
cytotoxic T cells to specific antigen stimulation in vitro, or to
non-specific stimulation such as PMA+Ionomycin.
The BD FastImmune™ Human IFN-γ/IL-4 Kit (Cat. No.
340456) enables rapid characterization of activated cells
expressing cytokines.
Intracellular flow cytometry offers distinct advantages over
classical methods for the detection of cytokines secreted
by T cells and other cells. It allows for the analysis of
cytokines and other inflammatory mediators produced by
multiple, phenotypically identified subpopulations within
a heterogeneous sample. It can determine whether the
cytokine production by an activated cell population is the
result of a few cells producing large amounts of cytokine or
a large cell population producing small quantities. Finally, it
can easily measure multiple cytokines simultaneously for an
individual cell.
Since cytokines typically are secreted proteins, they must
first be trapped inside the cell using a protein transport
inhibitor. The best choice of transport inhibitor varies by
cytokine and by species. Once trapped, most cytokines are
relatively accessible using the gentle BD Cytofix/Cytoperm
fixation and permeabilization solution.

BD Pharmingen Human Th1/Th2/Th17 Phenotyping Kit Clone Quantity Number of Tests Cat. No.
Human CD4 PerCP-Cy5.5 SK3
Human IL-17A PE N49-653
1 mL
Human IFN-γ FITC B27
Human IL-4 APC MP4-25D2 50 tests 560751
BD Cytofix™ Fixation Buffer 100 mL
BD Perm/Wash Buffer 25 mL
BD GolgiStop™ Protein Transport Inhibitor 0.7 mL

BD FastImmune Human IFN-γ/CD69/CD8/CD3 Kit Clone Quantity Number of Tests Cat. No.
Human IFN-γ FITC 25723.11
Human CD69 PE L78
1 mL 50 tests 346048
Human CD8 PerCP-Cy5.5 SK1
Human CD3 APC SK7

BD FastImmune Human IFN-γ/IL-4 Kit Clone Quantity Number of Tests Cat. No.
Human IFN-γ FITC 25723.11
1 mL 50 tests 340456
Human IL-4 PE 3010.211

10
 IFN-γ Production by Cytotoxic T Cells Cytokine Production by Stimulated Naïve T Cells
A C12 Unstimulated B C12 Unstimulated C D07 PMA+Ionomycin Unstimulated PMA+Ionomycin
Gate: (Lymphocytes in all) Gate: (CD3+8+ in (Lymphocytes in all)) Gate: (CD3+8+ in (Lymphocytes in all))
A05 Unstimulated A06 PMA + Ionomycin
10 6.4

10 6.2

10 6.2
CD69+ CD69+IFN+ CD69+ CD69+IFN+ Gate: (P1 in (R1 in all)) Gate: (P1 in (R1 in all))

10 6

10 6
1.5% 0.1% 27.4% 68.3%
Q1-UL Q1-UR Q1-UL Q1-UR
CD3+8+ 0.1% 0.0% 32.0% 0.3%

10 5

10 5
10 5

37.3%

10 5

10 5
FL3 CD8 PerCP Cy5.5-A

10 4

10 4
10 4

FL2 CD69 PE-A

FL2 CD69 PE-A

10 4

10 4
FL2 IL-17A PE-A

FL2 IL-17A PE-A

10 3

10 3
10 3

10 3

10 3
10 2

10 2
10 2

Q3-LL IFN+ Q3-LL IFN+

10 2

10 2
97.9% 0.5% 1.0% 3.3%
10 1

10 1

10 1
Q1-LL Q1-LR Q1-LL Q1-LR
10 1 10 2 10 3 10 4 10 5 10 6.2 10 1 10 2 10 3 10 4 10 5 10 6.2 10 1 10 2 10 3 10 4 10 5 10 6.2 99.8% 0.1% 64.5% 3.2%

10 1

10 1
FL4 CD3 APC-A FL1 IFN gamma FITC-A FL1 IFN gamma FITC-A
10 1 10 2 10 3 10 4 10 5 10 6 10 1 10 2 10 3 10 4 10 5 10 6
FL4 IL-4 APC-A FL4 IL-4 APC-A

A05 Unstimulated A06 PMA + Ionomycin

BD FastImmune IFN-γ/CD69/CD8/CD3 Kit (Cat. No. 346048) analysis Gate: (P1 in (R1 in all)) Gate: (P1 in (R1 in all))

10 6

10 6
Q2-UL Q2-UR Q2-UL Q2-UR
on the BD Accuri C6. 0.2% 0.0% 34.6% 0.4%

Immunology
Human whole blood was drawn into heparinized tubes and

10 5

10 5
FL1 IFN gamma FITC-A

FL1 IFN gamma FITC-A

stimulated with PMA (10 ng/mL) and Ionomycin (1 µg/mL) for 5

10 4

10 4
hours at 37°C in the presence of 10 ng/mL of Brefeldin A protein
transport inhibitor (BD GolgiPlug™, Cat. No. 555029). Samples

10 3

10 3
were harvested, fixed, lysed, permeabilized, and stained according Q2-LL
99.7%
Q2-LR
0.1%
Q2-LL
61.9%
Q2-LR
3.1%

10 2

10 2
to the kit procedure. They were acquired on a BD Accuri C6 flow 10 1 10 2 10 3 10 4
FL4 IL-4 APC-A
10 5 10 6 10 1 10 2 10 3 10 4
FL4 IL-4 APC-A
10 5 10 6

cytometer using the kit template and analyzed using BD Accuri C6

software, gating on lymphocytes using light scatter and then on BD Pharmingen Human Th1/Th2/Th17 Phenotyping Kit (Cat. No.
CD3+CD8+ cytotoxic T cells (A). Results: Compared to unstimulated 560751) analysis on the BD Accuri C6.
controls (B), stimulated cells (C) were more likely to produce high Purified human PBMCs were stimulated with PMA/Ionomycin (at 50
levels of the activation marker CD69. Most of these CD69+ cells ng/mL and 1 µg/mL, respectively) in the presence of BD GolgiStop
expressed IFN-γ as well. Data is representative of five donors. protein transport inhibitor (provided in the kit or Cat. No. 554724)
for 5 hours at 37°C. Cells were stained according to the kit
procedure, acquired on a BD Accuri C6 flow cytometer using the
IFN-γ and IL-4 Production by Activated T Cells
kit template, and analyzed using BD Accuri C6 software. Results:
A B C
B04 1009 Unstim Cocktail
Gate: Lymphocytes
B04 1009 Unstim Cocktail
Gate: (CD3+ in Lymphocytes)
B06 1009 Stim Cocktail
Gate: (CD3+ in Lymphocytes) Density plots (gated on CD4+ lymphocytes) show that stimulated
,500

10 5.9

10 5.9

Q1-UL
0.0%
Q1-UR
0.0%
Q1-UL
0.4%
Q1-UR
0.3%
cells (right) were more likely than unstimulated controls (left) to
10 5

10 5

produce high levels of IFN-γ, IL-4, and IL-17A as they differentiate

1,000

into Th1, Th2, and Th17 helper T cells, respectively.

10 4

10 4
FL2 IL4 PE-A

FL2 IL4 PE-A

Count

10 3

10 3
500

CD3+
77.4%
10 2

10 2

Q1-LL Q1-LR Q1-LL Q1-LR

100.0% 0.0% 67.4% 31.9%
10 1

10 1
0

10 1 10 2 10 3 10 4 10 5 10 6 10 7.2 10 1 10 2 10 3 10 4 10 5 10 6 10 6.8 10 1 10 2 10 3 10 4 10 5 10 6 10 6.8

FL4 CD3 APC-A FL1 IFN gamma FITC-A FL1 IFN gamma FITC-A

BD FastImmune IFN-γ/IL-4 Kit (Cat. No. 340456) analysis on the

BD Accuri C6.
Human lysed whole blood was activated with PMA/Ionomycin (at
50 ng/mL and 1 μg/mL, respectively) in the presence of Brefeldin
A protein transport inhibitor (BD GolgiPlug, Cat. No. 555029) for 4
hours at 37°C. Cells were washed, fixed, and permeabilized using the
BD Cytofix/Cytoperm fixation/permeabilization solution kit procedure
(Cat. No. 554714). After two washes with BD Perm/Wash buffer, the
cells were stained according to the kit procedure and with CD3 APC
(Cat. No. 555335) to identify T cells. They were acquired on a
BD Accuri C6 flow cytometer using the kit template and analyzed
using BD Accuri C6 software, gating on lymphocytes using
light scatter and then on CD3+ T cells (A). Results: Compared to
unstimulated controls (B), stimulated cells (C) were more likely to
produce high levels of IFN-γ (lower-right quadrant). Production of
high levels of IL-4 was rare (upper-left and upper-right quadrants),
but was also more common in stimulated than in unstimulated cells.

11
 Immunology: BD human Treg kits, protocols, and software templates
for the BD Accuri C6 simplify the detection and
Regulatory T Cells characterization of regulatory T cells (Tregs) using
intracellular and surface markers. The kits include
antibodies for key Treg markers such as FoxP3, CD4,
CD25, and CD127, along with buffer systems and
other reagents needed for acquisition and analysis.
The panels are compatible with other markers to
provide a base for Treg studies.

The BD Pharmingen™ FoxP3 Staining Kit (Cat. No. 560132)
provides two different analysis strategies for identifying Treg
populations, using FoxP3 expression in combination with CD4
and CD25.
The BD Pharmingen™ Human Regulatory T-Cell Cocktail (Cat.
No. 560249) is a one-step, premixed cocktail for convenient,
optimized analysis of Treg populations using surface markers,
without the need to permeabilize cells.
The BD Pharmingen™ Human Th17/Treg Phenotyping Kit
(Cat. No. 560762) can identify both Th17 and Tregs from a
single sample, and includes optimized reagents necessary for
successful intracellular staining.
Regulatory T cells, which suppress the function of other T cells,
play an important role in maintaining immune homeostasis.
The transcription factor FoxP3 is the classic marker for Tregs.
Because FoxP3 staining requires fixation and permeabilization
of cells, the cells cannot be used for further experiments.
However, the surface marker CD127 is negatively correlated
with FoxP3 and, when combined with CD4 and CD25 (as in the
BD Pharmingen Human Regulatory T-Cell Cocktail), enables
the identification of Tregs without permeabilization.
The discovery of FoxP3 has led to the characterization of
different types of Tregs. For example, natural Tregs (nTregs)
emerge from the thymus “naturally” expressing high levels of
FoxP3. In contrast, adaptive or inducible Tregs (iTregs) express
FoxP3 only after antigenic stimulation in the presence of
cognate antigen and specialized immunoregulatory cytokines.
iTregs are reported to be more plastic, with the ability to
convert to other T-cell subtypes such as Th1 and Th17, which
could undermine their eventual therapeutic value. All three
Treg kits can be used with antibodies to other markers such as
CD45RA to study Treg plasticity.

BD Pharmingen FoxP3 Staining Kit Clone Quantity Number of Tests Cat. No.
Human FoxP3 Alexa Fluor® 647 259D/C7 1 vial
Human CD4 FITC RPA-T4 1 vial
Human CD25 PE M-A251 1 vial 100 tests 560132
Human FoxP3 Buffer A (10X) 25 mL
Human FoxP3 Buffer A (50X) 100 mL

BD Pharmingen Human Th17/Treg Phenotyping Kit Quantity Number of Tests Cat. No.
Human CD4 PerCP-Cy5.5
Human IL-17 PE 1 mL
Human FoxP3 Alexa Fluor® 647
50 tests 560762
Human FoxP3 Buffer A (10X) 25 mL
Human FoxP3 Buffer A (50X) 100 mL
BD GolgiStop Protein Transport Inhibitor (containing monensin) 0.7 mL

BD Pharmingen Regulatory T-Cell Cocktail Clone Quantity Number of Tests Cat. No.
Human CD4 FITC SK3
Human CD25 PE-Cy™7 2A3 20 µL/test 50 tests 560249
Human CD127 Alexa Fluor® 647 hIL-7R-M21

All kits and their associated software templates are available on the BD Biosciences website.

12
 Identifying Tregs Using FoxP3 Distinguishing Tregs Using CD127
A A05 FoxP3 staining kie
Gate: (CD4 in (Lymphs in all))
B A05 FoxP3 staining kit
Gate: (CD25 in (Lymphs in all))
A A06 Donor_1060 Cocktail
Gate: (CD4 positives in Lymphocytes)
B A06 Donor_1060 Cocktail
Gate: (Non-Treg in (CD4 positives in Lymphocytes))
C A06 Donor_1060 Cocktail
Gate: (Tregs in (CD4 positives in Lymphocytes))

10 5.9

750
10 6.2

10 5.2

70
Q7-UL Q7-UR Q6-UL Q6-UR

FL4 CD127 Alexa Fluor® 647-A

18.0% 8.7% 0.6% 25.7%

FL4 FoxP3 Alexa Fluor® 647-A

60
10 5

600
Non-Treg
10 5

75.9%

10 4
10 4
Tregs
10 4

40
6.0%

400
FL2 CD 25 PE-A

Count

Count
10 3
10 3

10 3

200

20
10 2
10 2

Q7-LL Q7-LR Q6-LL Q6-LR

71.9% 1.4% 21.1% 52.6%

10 1
10 1

10 2

0
10 2 10 3 10 4 10 5.2 10 2 10 3 10 4 10 5 10 6.2 10 1 10 2 10 3 10 4 10 5 10 5.9 10 0 10 1 10 2 10 3 10 4 10 5 10 5.9 10 0 10 1 10 2 10 3 10 4 10 5 10 5.9

FL4 FoxP3 Alexa Fluor® 647-A FL1 CD4 FITC-A FL3 CD25 PE-Cy7-A FL2 FoxP3 PE-A FL2 FoxP3 PE-A

BD Pharmingen FoxP3 Staining Kit (Cat. BD Pharmingen Human Regulatory T-Cell Cocktail (Cat. No. 560249)
No. 560132) analysis on the BD Accuri C6. analysis on the BD Accuri C6.

Immunology
Human PBMCs were stained with CD4 FITC Human PBMCs were stained according to the kit procedure.
and CD25 PE, fixed, permeabilized, and The cells were then fixed, lysed, and permeabilized using the
stained for intracellular content with FoxP3 BD Pharmingen™ Human Transcription Factor Buffer Set
Alexa Fluor® 647 according to the (Cat. No. 562574) and stained with PE-conjugated anti-human
kit procedure. Samples were collected on BD Pharmingen™ FoxP3 monoclonal antibody (Cat. No. 560082).
the BD Accuri C6 flow cytometer using Samples were collected on the BD Accuri C6 flow cytometer using
the kit template and analyzed using the kit template and analyzed using BD Accuri C6 software. CD4+
BD Accuri C6 software. Results: A. Gating on lymphocytes were identified and gated by light scatter profile and
CD4+ lymphocytes, Tregs are CD25+FoxP3+. fluorescence (data not shown). Results: A. A CD25 vs CD127 plot
B. Gating on CD25+ lymphocytes, Tregs are was used to identify CD25brightCD127dim Tregs and non-Treg CD4+
CD4+FoxP3+. Both plots depict equivalent cells. B, C. Tregs identified in Panel A were validated using FoxP3
percentages of Tregs. staining. CD4+ cells identified as Tregs (C) expressed higher levels of
FoxP3 than did non-Tregs (B).

Distinguishing Th17 Cells and Tregs

in a Single Sample
A B09 Unstimulated B C03 PMA Ionomycin stim
Gate: (CD4+ in (Lymphocytes in all)) Gate: (CD4+ in (Lymphocytes in all))
10 5.3
10 5.3

Th17 Q7-UR Th17 Q7-UR

0.1% 0.0% 1.7% 0.2%
FL2 IL-17 PE-A

FL2 IL-17 PE-A

10 4
10 4

10 3
10 3

10 2
10 2

Q7-LL Treg Q7-LL Treg

92.3% 7.5% 89.1% 9.0%
10 1.5
10 1.5

10 1.2 10 2 10 3 10 4 10 5.1 10 1.2 10 2 10 3 10 4 10 5.1

FL4 FoxP3 Alexa Fluor® 647-A FL4 FoxP3 Alexa Fluor® 647-A

BD Pharmingen Human Th17/Treg

Phenotyping Kit (Cat. No. 560762) analysis
on the BD Accuri C6.
Human PBMCs were either unstimulated
or stimulated with PMA and Ionomycin for
4 hours in the presence of BD GolgiStop
protein transport inhibitor (included in
the kit or Cat. No. 554724). The cells were
fixed, permeabilized, and stained with
the antibody cocktail according to the kit
procedure. Samples were collected on the
BD Accuri C6 flow cytometer using the kit
template and analyzed using BD Accuri C6
software. CD4+ lymphocytes were identified
and gated by light scatter profile and
fluorescence (data not shown). Results:
Compared to unstimulated cells (A), more
PBMCs stimulated with PMA and Ionomycin
(B) expressed IL-17, and some expressed
FoxP3 as well.

13
 Stem Cells: BD pluripotent stem cell kits, protocols, and
software templates for the BD Accuri C6 simplify the
Pluripotent Stem Cells characterization of pluripotent stem cells and their
derivatives. The kits support studies involving key
pluripotency and differentiation markers such as
SSEA-1, SSEA-3, SSEA-4, TRA-1-81, Nanog, Oct3/4,
and Sox2. They also include buffer systems and
controls needed for acquisition and analysis, and are
compatible with other markers to provide a base for
studies of stem cell pluripotency and differentiation.

The BD Stemflow™ Human Pluripotent Stem Cell Sorting
and Analysis Kit (Cat. No. 560461), which includes
antibodies to TRA-1-81, SSEA-1, and SSEA-3, provides a
comprehensive system for characterizing pluripotent stem
cells using flow cytometry.
The BD Stemflow™ Human Pluripotent Stem Cell
Transcription Factor Analysis Kit (Cat. No. 560589) can
simultaneously measure expression of the stem cell master
regulators—Nanog, Oct3/4, and Sox2—in heterogeneous
stem cell populations.
The BD Stemflow™ Human and Mouse Pluripotent Stem
Cell Analysis Kit (Cat. No. 560477) enables reliable, in-depth
characterization of cellular pluripotency and differentiation
state in heterogeneous human or mouse stem cell cultures
using antibodies to both cell surface (SSEA-1, SSEA-4) and
intracellular markers (Oct 3/4).
Flow cytometry offers an easy, rapid, and quantitative
method of analyzing established markers of stem cell
pluripotency and differentiation. Researchers can examine
the cells’ transcriptional and surface profile to correlate
with pluripotency, study their differentiation states, or
scrutinize heterogeneous cell populations to identify culture
dynamics.
Pluripotent stem cells differentiate into the three
primary germ layers and into differentiated tissue, each
characterized by certain cell surface proteins and/or
intracellular transcription factors. Antibodies to these
markers can be combined in many ways to monitor the cells’
changing expression patterns. Analysis based on cell surface
markers can preserve cell viability for use in additional
experiments.

BD Stemflow Human Pluripotent Stem Cell Sorting Clone Quantity Number of Tests Cat. No.
and Analysis Kit
TRA-1-81 Alexa Fluor® 647 TRA-1-81 1.5 mL
SSEA-1 FITC MC480 1.5 mL
50 tests 560461
SSEA-3 PE MC631 1.5 mL
Controls and compensation particles as detailed in kit manual

BD Stemflow Human Pluripotent Stem Cell Transcrip- Clone Quantity Number of Tests Cat. No.
tion Factor Analysis Kit
Nanog PE N31-355 1.5 mL
Oct3/4 PerCP-Cy5.5 40/Oct-3 1.5 mL
50 tests 560589
Sox2 Alexa Fluor® 647 245610 1.5 mL
Controls and compensation particles as detailed in kit manual

BD Stemflow Human and Mouse Pluripotent Stem Clone Quantity Number of Tests Cat. No.
Cell Analysis Kit
Oct3/4 PerCP-Cy5.5 40/Oct-3 1.5 mL
SSEA-4 Alexa Fluor® 647 MC813 1.5 mL
50 tests 560477
SSEA-1 PE MC480 1.5 mL
Controls and compensation particles as detailed in kit manual

All kits and their associated software templates are available on the BD Biosciences website.

14
 Analyzing Stem Cells for Key Transcription Factors Characterizing Stem Cell Pluripotency and DIfferentiation State
A F02 08_Ab stained B F02 08_Ab stained C F02 08_Ab stained A D02 05_Ab stained B D02 05_Ab stained C D02 05_Ab stained
Gate: (P1 in all) Gate: (P1 in all) Gate: (P1 in all) Gate: (P1 in all) Gate: (P1 in all) Gate: (P1 in all)

10 6

10 6

10 6
10 6

10 6

10 6
Q8-UL Q8-UR Q9-UL Q9-UR Q10-UL Q10-UR Q1-UL Q1-UR Q2-UL Q2-UR Q3-UL Q3-UR
0.6% 95.1% 6.9% 88.7% 7.0% 90.2% 0.2% 1.0% 0.0% 1.5% 0.0% 75.0%

10 5
10 5

10 5
10 5

10 5

10 5

FL3 Oct3-4 PerCP-Cy5.5-A

FL3 Oct3-4 PerCP-Cy5.5-A
FL2 Nanog PE-A

FL2 Nanog PE-A

FL1 SSEA-1 PE-A

FL1 SSEA-1 PE-A

10 4
10 4

10 4

10 4

10 4

10 4
10 3
Q8-LL Q8-LR Q9-LL Q9-LR Q10-LL Q10-LR Q1-LL Q1-LR Q2-LL Q2-LR Q3-LL Q3-LR
2.6% 1.7% 2.4% 2.0% 2.3% 0.5% 27.5% 71.4% 0.0% 98.5% 0.0% 25.0%
10 2.7

10 2.7

10 2.7

10 2.7

10 2.5

10 2.7
10 2.7 10 4 10 5 10 6 10 2.6 10 3 10 4 10 5 10 6 10 2.6 10 3 10 4 10 5 10 6 10 2 10 3 10 4 10 5 10 6 10 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7
FL3 Oct3-4 PerCP-Cy5.5-A FL4 Sox2 Alexa 647-A FL4 Sox2 Alexa 647-A FL3 Oct3-4 PerCP-Cy5.5-A FL4 SSEA-4 Alexa 647-A FL4 SSEA-4 Alexa 647-A

BD Stemflow Human Pluripotent Stem Cell Transcription Factor BD Stemflow Human and Mouse Pluripotent Stem Cell Analysis Kit
Analysis Kit (Cat. No. 560589) analysis on the BD Accuri C6. (Cat. No. 560477) analysis on the BD Accuri C6.
H9 human embryonic stem cells (hESCs) were disassociated using H9 hESCs were disassociated using BD Accutase Cell Detachment
BD™ Accutase Cell Detachment Solution (Cat. No. 561527), stained Solution (Cat. No. 561527), stained according to kit instructions,
according to kit instructions, and acquired on a BD Accuri C6 and acquired on a BD Accuri C6 flow cytometer using the kit
flow cytometer using the kit template. Cells were gated on light template. Cells were gated on light scatter properties of H9 hESCs
scatter properties of H9 hESCs andA analyzed
B03 Kit
Gate: (P3 in all)
for expression of core and analyzed for expression of key pluripotency surface markers
pluripotency transcription factors using
Q4-ULBD Accuri Q4-UR C6 software. and transcription factors using BD Accuri C6 software. Results: Most
7.2
107.2
10

88.8% 2.7%
Results: Most analyzed cells expressed the core pluripotency analyzed cells expressed the positive pluripotency surface marker
106
10 6

transcription factors Nanog, Oct3/4, and Sox2. SSEA-4 and the pluripotency transcription factor Oct3/4, while few
105
10 5
SSEA-3-A
FL2 PE SSEA-3-A

expressed the negative pluripotency marker (positive differentiation

104
10 4

marker) SSEA-1.
103
10

Stem Cells
10 2

Q4-LL Q4-LR
7.9% 0.6%
10 1

Analyzing Stem Cells for Key Pluripotency Markers

FL1 FITC SSEA-1-A
10 1 10 2 10 3 10 4 10 5 10 6 107.2
10

A B03 Kit B B03 Kit C B03 Kit

Gate: (P3 in all) Gate: (P3 in all) Gate: (P3 in all)
10 7.2

10 7.2

10 7.2

Q4-UL Q4-UR Q5-UL Q5-UR Q6-UL Q6-UR

Q6-UR
88.8% 2.7% 81.6% 5.1% 5.2% 84.0%
84.0%
10 6

10 6

10 6
FL4 Alexa 647 TRA-1-81-A

FL4 Alexa 647 TRA-1-81-A

10 5

10 5

10 5
FL2 PE SSEA-3-A
10 4

10 4

10 4
10 3

103
10
10

10
10 2

102
10
10

10

Q4-LL Q4-LR Q5-LL Q5-LR Q6-LL Q6-LR

Q6-LR
FL4

FL4

7.9% 0.6% 12.2% 1.1% 1.7% 9.1%

9.1%
10 1

10 1

1
1

101
10

10

10 1 10 2 10 3 10 4 10 5 10 6 10 7.2 101
10
1 102
10 103
10 10 4 10 5 10 6 10
7.2 10 1 10 2 10
10
3 10
10
4 5
10 5
10 6
10 6
10 7.2
10 7.2
10
FL1 FITC SSEA-1-A FL1 FITC SSEA-1-A FL2 PE SSEA-3-A
SSEA-3-A

B03 Kit C B03 Kit

Gate: (P3 in all) Gate: (P3 in all)
BD Stemflow Human Pluripotent
Q6-UR Stem Cell Sorting and Analysis Kit
10 7.2

Q5-UL Q5-UR Q6-UL

81.6% 5.1% 5.2% 84.0%
(Cat. No. 560461) analysis on the BD Accuri C6.
10 6

H9 hESCs were disassociated using BD Accutase Cell Detachment

FL4 Alexa 647 TRA-1-81-A
10 5

Solution (Cat. No. 561527), stained according to kit instructions, and

10 4

acquired on a BD Accuri C6 flow cytometer using the kit template.

10 3

CellsQ5-LR
were gated on light scatter properties of H9 hESCs and analyzed
10 2

Q5-LL Q6-LL Q6-LR

12.2%
for5 expression
1.1%
of 2key3 pluripotency
1.7% 9.1%
surface markers using BD Accuri C6
10 1

1 2
10 3
10 4 10 6 7.2 1 10 4 5
10 6 7.210 10 10 10 10 10 10 10

software. Results: Most

FL1 FITC SSEA-1-A
analyzed cells expressed positive pluripotency
FL2 PE SSEA-3-A

surface markers SSEA-3 and TRA-1-81, while few expressed the

negative pluripotency marker (positive differentiation marker) SSEA-1.

15
 Bead-Based Assays: Bead-based flow cytometric immunoassays are
powerful methods of quantifying proteins because
BD Cytometric Bead they allow researchers to multiplex many analytes
with very little sample.
Array Kits

BD CBA Kit Bead Resolution There are two ways to use BD CBA: preconfigured kits
(described here) and fully configurable flex sets (described
200

further at bdbiosciences.com/go/cba/).
150

BD CBA kits provide preconfigured panels of 3–7 analytes

for ultimate ease of use. For example, the BD™ CBA
100
Count

Human Th1/Th2/Th7 Cytokine Kit can quantitate IL-2, IL-4,

50

IL-6, IL-10, TNF, IFN-γ, and IL-17A simultaneously. Other

0

10 3 10 4 10 5
FL4 675/25
10 6 10 7.2
kits include panels of human cytokines, chemokines, and
anaphylatoxins; mouse cytokines and immunoglobulins; and
10 6.1

BD CBA kit bead resolution on the

BD Accuri C6.
non-human primate cytokines.
The beads used in BD CBA kits are resolved On the BD Accuri C6, the beads are excited by the red laser
10 5
FL3 780/60

clearly in the FL4 detector of the BD Accuri

and detected in FL4, while the PE reporter is excited by the
C6 using the standard 675/25 filter.
blue laser and detected in FL2. A BD Accuri C6 software
10 4
10 3.6

10 4 10 5 10 6 10 6.8 template simplifies setup and acquisition. Results can be

FL4 675/25
displayed in FCAP Array™ software by bead (graphing one
analyte across all samples) or by sample (graphing one
sample across all analytes).

Analyze by Concentration
Beads Sample Wash Flow Cytometry

F
PE MFI
NIR

A
Detector Antibodies
B
MFI

Red
E

Concentration (pg/mL)
Standard Curve

16
A BD™ Cytometric Bead Array (CBA) assay contains a cocktail of beads bound with specific capture antibodies
that differ slightly in fluorescence intensity. The beads are mixed with samples along with PE-labeled detection
antibodies and run on a BD Accuri C6. BD CBA can analyze 300 beads per protein—the equivalent of 300 ELISA
wells—for up to 30 cytokines simultaneously. In essence, BD CBA is like running multiple ELISAs at once using
flow cytometry.

BD CBA Kit Bead Resolution

A B

BD CBA kit analysis by analyte or sample.

Human PBMCs were cultured for several

Bead-Based Assays
days with plate-bound anti-CD3, soluble
anti-CD27, IL-2, and IL-4. Cells were
Brand Kit Cat. No.
stimulated with PMA+Ionomycin for several
hours before collecting culture supernatants. BD™ Cytometric Bead Array Human Anaphylatoxin Kit 561418

Samples were prepared and stained with the BD™ Cytometric Bead Array Human Chemokine Kit 552990
BD CBA Human Th1/Th2/Th17 Cytokine Kit BD™ Cytometric Bead Array Human Inflammatory Cytokine Kit 551811
(Cat. No. 560484), acquired on a BD Accuri BD™ Cytometric Bead Array Human Inflammatory Cytokine Lyophilized Standard 552932
C6 using the BD CBA Kit Accuri template,
BD™ Cytometric Bead Array Human Th1/Th2 Cytokine Kit II 551809
and analyzed using FCAP Array software
BD™ Cytometric Bead Array Human Th1/Th2 Cytokine Kit 550749
v3.0.1. A. Bar chart of human IFN-γ levels for
all standards and test samples. B. Bar chart BD™ Cytometric Bead Array Human Th1/Th2 Cytokine Lyophilized Standard 551810

of all seven cytokine levels for one sample. BD™ Cytometric Bead Array Human Th1/Th2 Cytokine Standards 561666
BD™ Cytometric Bead Array Human Th1/Th2/Th17 Kit 560484
BD™ Cytometric Bead Array Mouse Immunoglobulin Isotyping Kit 550026
BD™ Cytometric Bead Array Mouse Inflammation Kit 552364
BD™ Cytometric Bead Array Mouse Inflammation Lyophilized Standard 620280
BD™ Cytometric Bead Array Mouse Th1/Th2 Cytokine Kit 551287
BD™ Cytometric Bead Array Mouse Th1/Th2 Cytokine Lyophilized Standard 552967
BD™ Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine Kit 560485
BD™ Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine Standards 561665
BD™ Cytometric Bead Array Non-human Primate Th1/Th2 Cytokine Kit 557800
Required Software
FCAP Array Software v3.0 (Microsoft® Windows® 7, Windows Vista®, XP) 652099

All kits, a product information sheet, and the BD™ CBA Kit Accuri
Template are available on the BD Biosciences website.

17
 Microbiology: The BD Accuri™ C6 Eawag Water Quality Template2
simplifies the enumeration of intact and damaged
Water Quality bacteria in drinking water samples on the BD Accuri C6.
The Eawag protocol involves co-staining the samples
with SYBR® Green I and (optionally) propidium iodide
(PI). The template exploits the ability of the BD Accuri
C6 to calculate absolute cell counts and concentrations
automatically.

Enumerating Intact and Damaged Bacteria in Drinking Water Using nucleic acid dyes, flow cytometry can quantitate
microbes and discriminate them from debris much more
A 105
LNA: low nucleic acid content cells
B 105 C 105 rapidly than plate methods. SYBR® Green I, which
damaged cells Water sample 1
Red fluorescence (670 nm)

Red fluorescence (670 nm)

HNA: high nucleic acid content cells

Water sample 2 preferentially stains double-stranded DNA, is well suited
Bacteria cell count

Water sample 3
104 background 104 104
for staining bacteria in water samples for flow cytometric
103 HNA 103 103
analysis. PI can be added to differentiate live and damaged
102 LNA 102 102
bacterial cells. PI is impermeable to healthy cells with
bacteria intact cells
intact membranes, but permeates cells with compromised
membranes such as dead cells.
103 103 103 103 103 103 103 104 105
Green fluorescence (533 nm) Green fluorescence (533 nm) Green fluorescence (533 nm)

Water quality analysis using the Eawag water quality template on For a detailed discussion of the use of the Eawag template
the BD Accuri C6. and protocol for water quality research, see the
Drinking water samples were stained according to the Eawag
BD Biosciences white paper, Assessing Water Quality with
protocol, acquired on a BD Accuri C6 using the Eawag water quality
template, and analyzed using BD Accuri C6 software. A. When a
the BD Accuri™ C6 Flow Cytometer (January 2013), available
sample is stained with SYBR® Green I, all bacteria appear within the at bdbiosciences.com.
template’s single, fixed gate, while noise and debris are excluded.
2. The water quality template and staining protocol were developed in collaboration with
B. When the sample is co-stained with SYBR® Green I and Eawag, the Swiss Federal Institute of Aquatic Science and Technology.
propidium iodide (PI), damaged bacteria are shifted out of the gate,
leaving only viable bacteria within. C. Each water sample generates
a unique flow cytometric fingerprint.

Data Courtesy of Frederik Hammes, Eawag Department of Environmental Microbiology,

Dübendorf, Switzerland.
Microbiology

BD Accuri C6 Eawag Water Quality Template (zip file)

2012 BD Template_Eawag_24 tube rack.c6t file
2012 BD Template_Eawag_96 well plate.c6t file
Eawag Water Quality Template ReadMe.doc file

The template and product information sheet are

available on the BD Biosciences website.

18
Instrument Setup: BD Accuri™ Spherotech Validation Beads contain a
mixture of fluorochromes that are spectrally similar to
Validation Beads many of the fluorochromes used in flow cytometry.
Due to the unique design of the BD Accuri C6, there
is no need to use the beads to align the system or to
adjust voltages. Instead, you can use the beads to
validate the linearity, sensitivity, and resolution of the
cytometer detectors at the beginning of each workday.
Two BD Accuri C6 software templates make daily
validation easy and convenient.

Validating Performance of the BD Accuri C6 BD Accuri™ Spherotech 8-peak Validation Beads (Cat. No.
653144) are used to validate performance of the FL1 (FITC),
FL2 (PE), and FL3 (PE-Cy™5) detectors.
6,400

6,400

6,400

6,400

BD Accuri™ Spherotech 6-peak Validation Beads (Cat. No.

4,000

4,000

4,000

4,000
Counts
2,000 Counts

Counts
2,000 Counts

653145) are used to validate performance of the FL4 (APC)

2,000

2,000

detector.

For detailed procedural and troubleshooting steps to use in

0

101 10
101 2 10
102 3 10
103 4 10
104 5 10
105 6 10
1067.2 107.2 101 10
101 2 10
102 3 10
103 4 10
104 5 10
105 6 10
1067.2 107.2
FL1-HFL1-H FL2-HFL2-H
validating the BD Accuri C6, see Chapter 2 of the BD Accuri™
6,400

6,400

6,400

6,400

C6 Software User Guide, available at bdbiosciences.com.

4,000

4,000

4,000

4,000
Counts
2,000 Counts

Counts
2,000 Counts
2,000

2,000
0

101 10
101 2 10
102 3 10
103 4 10
104 5 10
105 6 10
1067.2 107.2 101 10
101 2 10
102 3 10
103 4 10
104 5 10
105 6 10
1067.2 107.2
FL3-HFL3-H FL4-HFL4-H

Analysis of BD Accuri Spherotech Validation Beads (Cat. Nos. 653144

and 653145) on the BD Accuri C6.
8-Peak beads were used to validate performance of the FL1, FL2,
and FL3 detectors, while 6-Peak beads were used to validate the FL4
detector. Software templates were used to streamline acquisition
and analysis.

Description Particle size, µm Size Cat. No.

BD Accuri Spherotech Validation Beads (8 peaks) 3.0–3.4 4 mL 653144
BD Accuri Spherotech Validation Beads (6 peaks) 3.0–3.4 2 x 4 mL 653145

The templates and the BD Accuri™ C6 Software User Guide are available on the
BD Biosciences website.
Instrument Setup

19
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21
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Office locations are available on our websites.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD flow cytometers are Class 1 Laser Products.
Cy™ is a trademark of GE Healthcare. Cy™ dyes are subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and are made and sold under license from GE Healthcare only
for research and in vitro diagnostic use. Any other use requires a commercial sublicense from GE Healthcare, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.
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