Kits and Templates for the BD Accuri™ C6€¦ · A template is a predefined workspace that...

As Easy as Cell Analysis is Going to Get

Kits and Templates for the BD Accuri™ C6

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.

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

As easy as cell analysis is going to get

Reagent KitsBD 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 TemplatesFree 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.

2

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 C6The 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.

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).

Using Kits and TemplatesTemplate Home PageReagent 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 TemplatesOnce 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 templateSelect File > Open workspace or template. All settings are fully adjustable.

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BD Pharmingen Annexin V FITC Apoptosis Detection Kit II Quantity Number of Tests Cat. No.

Purified Recombinant Annexin V 100 μg

100 tests 556570FITC Annexin V 0.5 mL

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

100 tests 5597637-AAD 2.0 mL

10X Annexin V Binding Buffer 2.0 mL

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

PE Rabbit Anti-Active Caspase-3 20 μL/test

100 tests 550914BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution 65 mL

BD Perm/Wash™ Buffer (10X Solution) 65 mL

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

JC-1 50 mL100 tests 551302

10X Assay Buffer 60 mL

Cell Biology: Apoptosis

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.

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

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.

4

6,990,5072,000,0000 4,000,000

,595

,118

1,00

0,00

00

2,00

0,00

0SS

C-A

FSC-A

A05 DMSOGate: [No Gating]

P196.3%P196.3%

102.7 105.2103 104

1,00

050

00

Co

un

t

FL2 Active caspase-3 PE-A

A05 DMSOGate: (P1 in all)

V4-L97.0%

V4-R3.0%

V4-L97.0%

V4-R3.0%

102.7 105.2103 104

1,00

050

00

Co

un

t

FL2 Active caspase-3 PE-A

A06 CamptothecinGate: (P1 in all)

V4-L74.2%

V4-R25.8%

V4-L74.2%

V4-R25.8%

A B C

599,187 14,280,6075,000,000 10,000,000

5,29

2,81

32,

000,

000

04,

000,

000

SSC

-A

FSC-A

A02 K562 untreated JC-1Gate: [No Gating]

P184.8%P184.8%

103.7 107.2105 106103.

310

7.2

104

105

106

FL2

JC-1

-A

FL1 JC-1-A

A02 K562 untreated JC-1Gate: (P1 in all)

P515.0%

P483.0%

P515.0%

P483.0%

103.7 107.2105 106103.

310

7.2

104

105

106

FL2

JC-1

-A

FL1 JC-1-A

A03 K562 CCCP JC-1 Gate: (P1 in all)

P591.4%

P47.7%

P591.4%

P47.7%

A B C

0 10,067,2835,000,000

,695

,318

500,

000

01,

000,

000

SSC

-A

FSC-A

B02 DMSO FITC + PIGate: [No Gating]

P188.4%P188.4%

101 107.2102 103 104 105 106

101

107.

210

210

310

410

510

6FL

3 Pr

op

idiu

m Io

did

e-A

FL1 Annexin V FITC-A

B02 DMSO FITC + PIGate: (P1 in all)

Q5-UL0.1%

Dead11.3%

Live85.4%

Apoptotic3.2%

Q5-UL0.1%

Dead11.3%

Live85.4%

Apoptotic3.2%

0 10,067,2835,000,000

,695

,318

500,

000

01,

000,

000

SSC

-A

FSC-A

B03 Camptothecin FITC + PIGate: [No Gating]

P153.1%P153.1%

101 107.2102 103 104 105 106

101

107.

210

210

310

410

510

6FL

3 Pr

op

idiu

m Io

did

e-A

FL1 Annexin V FITC-A

B03 Camptothecin FITC + PIGate: (P1 in all)

Q5-UL0.1%

Dead9.3%

Live62.3%

Apoptotic28.3%

Q5-UL0.1%

Dead9.3%

Live62.3%

Apoptotic28.3%

Detecting Early-Stage Apoptosis Using Annexin V

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.

Detecting Mitochondrial Membrane Changes Using JC-1 Detecting Apoptosis Using Caspase Activation

BD MitoScreen (JC-1) Kit (Cat. No. 551302) analysis on the BD Accuri C6.K562 cells (human chronic myelogenous leukemia; ATCC CCL-243) were treated with 100 μM of CCCP (in DMSO) for 5 minutes at 37ºC to induce mitochondrial membranes to decouple. The cells were stained with JC-1 and washed according to the kit protocol, acquired on a BD Accuri C6 flow cytometer using the kit template, and analyzed using BD Accuri C6 software. Results: Compared to untreated controls (B), CCCP treatment (C) resulted in a shift in mitochondrial membrane potential (red to green). Because JC-1 staining patterns can vary for different cell types and experimental conditions, P4 and P5 gate boundaries should be adjusted according to the kit guidelines.

BD Pharmingen Caspase-3 Assay Kit (Cat. No. 550914) 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 permeabilized, fixed, and stained according to the kit protocol, acquired on a BD Accuri C6 flow cytometer using the kit template, and analyzed using BD Accuri C6 software. Results: Camptothecin treatment (C) resulted in an increase in active caspase-3 expression compared to the DMSO control (B), which was almost completely negative.

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5

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.

Cell Biology: Cell Cycle and DNA

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.

BD cell cycle/DNA kits, protocols, and software

templates for the BD Accuri C6 simplify the assessment

of cell cycle and DNA status.

6

105.3 106.6106105.

110

6.5

106

FL2

Pro

pid

ium

Iod

ide-

H

FL2 Propidium Iodide-A

A03 K562Gate: (P2 in all)

Singlets93.9%Singlets93.9%

A

400,000 1,300,0001,000,000

1,60

050

00

1,00

0C

ou

nt


A03 K562Gate: (Singlets in (P2 in all))

G0/G144.6%

S34.5%

G2/M21.3%

G0/G144.6%

S34.5%

G2/M21.3%

B

300,000 1,300,000500,000 1,000,000

050

020

040

0C

ou

nt


A05 PBMC + K562Gate: (Singlets in all)

PBMCs G0/G1

K562 G0/G1

PBMCs G0/G1MFI=396,087

K562 G0/G1 MFI=571,768

C

BD Cycletest Plus DNA Reagent Kit Quantity Number of Tests Cat. No.

Solution A: Trypsin in spermine tetrahydrochloride detergent buffer 10 mL

40 tests 340242Solution B: RNase A and trypsin inhibitor in spermine buffer 8 mL

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

50 tests559619 (FITC)552598 (APC)

DNase 5 x 300 μL

Fluorochrome-conjugated anti-BrdU antibody 1 x 65 μL

BD Cytofix/Cytoperm buffer 1 x 25 mL

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

0 120,00050,000

102.

710

710

410

510

6FL

1 B

rdU

FIT

C-A

FL3 7-AAD-A

A04 PI FITC BrdU + 7-AADGate: (P3 in all)

S6.8%

G0/G170.9%

G2/M4.0%

S6.8%

G0/G170.9%

G2/M4.0%

0 100,00050,000

103

107

104

105

106

FL4

Brd

U A

PC-A

FL3 7-AAD-A

A05 PI APC BrdU + 7-AADGate: (P3 in all)

G0/G169.6%

G2/M3.2%

S6.4%

G0/G169.6%

G2/M3.2%

S6.4%

A B

Identifying Cell Cycle Phases Using BrdU and 7-AAD Assessing Cell Cycle and Ploidy Using PI

BD Pharmingen FITC and APC BrdU Flow Kits (Cat. Nos. 559619 and 552598) analysis on the BD Accuri C6.Human peripheral blood mononuclear cells (PBMCs) were stimulated, expanded, restimulated, and labeled with 20 μM of BrdU during the final hour. After harvesting and staining the cells with 7-AAD and either FITC or APC anti-BrdU according to the kit protocol, samples were acquired on a BD Accuri C6 flow cytometer using the kit template, and analyzed using BD Accuri C6 software. Cell cycle phases are clearly distinguished 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.

BD Cycletest Plus DNA Reagent Kit (Cat. No. 340242) analysis on the BD Accuri C6.K562 leukemia cells (incorporating the Philadelphia translocation) were cultured and stained according to the kit protocol, acquired on a BD Accuri C6 flow cytometer using the kit template, and analyzed using BD Accuri C6 software. A. K562 cells were gated to exclude aggregates on a PI FL2-A vs PI FL2-H plot. B. A PI histogram of the gated K562 singlets distinguishes cells at the G0/G1, S, and G2+M cycle phases. Gating of cell cycle phases is approximate and can be refined using software analysis. C. Staining and analyzing normal PBMCs along with the K562 cells can quantify their aneuploidy by gating on their G0/G1 peaks. The ratio of the MFIs of the two peaks, called the DNA Index (DI), serves as a measure of aneuploidy—in this case 1.4.

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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.

7

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.

Immunology: Naïve/Memory T 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 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.

8

Imm

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101.4 105.4102 103 104 105101.

510

5.9

102

103

104

105

FL4

CD

4 A

PC-A

FL1 CD45RA FITC-A

C11Gate: (CD3+ in all)

Naive11.6%Naive11.6%

101.4 105.4102 103 104 105101.

510

5.9

102

103

104

105

FL4

CD

4 A

PC-A

FL2 CD45RO PE-A


Memory51.7%Memory51.7%

A

B

101.4 105.4102 103 104 105101.

510

5.9

102

103

104

105

FL4

CD

4 A

PC-A

FL1 CD45RA FITC-A


Naive11.6%Naive11.6%

101.4 105.4102 103 104 105101.

510

5.9

102

103

104

105

FL4

CD

4 A

PC-A

FL2 CD45RO PE-A


Memory51.7%Memory51.7%

A

B

102 104.7103 104

1,99

7,28

850

0,00

00

1,00

0,00

01,

500,

000

SSC

-A

FL3 CD4 PerCP Cy5.5-A

C06Gate: [No Gating]

CD4+6.9%CD4+6.9%

101.5 105.1102 103 104101.

910

4.8

103

104

FL4

CD

197

AF

647-

A

FL1 CD45RA FITC-A


Central Memory22.5%

Naive44.6%

Effector Memory31.6%

Q1-LR1.3%

Central Memory22.5%

Naive44.6%

Effector Memory31.6%

Q1-LR1.3%

A B

101 106.2102 103 104 105

101

106.

210

210

310

410

5FL

4 C

D4

APC

-A

FL3 CD3 PerCP-A

B06Gate: (CD3+ in all)

CD3+CD4+72.9%CD3+CD4+72.9%

101.3 105.2102 103 104101.

310

5.5

102

103

104

105

FL2

CD

62L

PE-A

FL1 CD45RA FITC-A

B06Gate: (CD3+CD4+ in (CD3+ in all))

Non-Activated Memory 52.4%

Naive25.5%

Activated Memory21.4%

Q1-LR0.7%

Non-ActivatedMemory 52.4%

Naive25.5%

Activated Memory21.4%

Q1-LR0.7%

A B

Differentiating Naïve vs Memory T Cells

Distinguishing Activated vs Non-Activated Memory Cells

Distinguishing Central vs Effector Memory Cells

BD Multitest Human CD45RA/CD45RO/CD3/CD4 Kit (Cat. No. 340571) analysis on the BD Accuri C6.Human peripheral blood was stained according to the kit procedure, acquired on a BD Accuri C6 flow cytometer using the kit template, and analyzed using BD Accuri C6 software with a gate set on CD3+ lymphocytes. Results: The CD3+ T cells were characterized as either (A) naïve (CD4+CD45RA+, blue) or (B) memory cells (CD4+CD45RO+, red).

BD Multitest Human CD45RA/CD62L/CD3/CD4 Kit (Cat. No. 340977) analysis on the BD Accuri C6.Human peripheral blood was stained according to the kit procedure, acquired on a BD Accuri C6 flow cytometer using the kit template, and analyzed using BD Accuri C6 software with a gate set on CD3+ lymphocytes. Results: A. An additional gate was drawn around the CD3+CD4+ T-cell population. B. The CD3+CD4+ T cells were characterized as either naïve (CD45RA+CD62L+, blue), non-activated memory (CD45RA–CD62L+, red), or activated memory cells (CD45RA–CD62L–, purple).

BD Pharmingen Human Naïve/Memory T Cell Panel (Cat. No. 561438) analysis on the BD Accuri C6.Human peripheral blood was stained according to the kit procedure, acquired on a BD Accuri C6 flow cytometer using the kit template, and analyzed using BD Accuri C6 software. (The optional drop-in reagent CD3 APC-H7 was not used.) Results: A. A gate was drawn around the CD4+ T-cell population. B. The CD4+ T cells were characterized as either naïve (CD45RA+CD197+, blue), central memory (CD45RA–CD197+, red), or effector memory cells (CD45RA–CD197–, purple).

BD Multitest Human CD45RA/CD45RO/Cd3/CD4 Kit Clone Quantity Number of Tests Cat. No.

Human CD3 PerCP SK7

20 μL/test 50 tests 340571Human CD4 APC SK3

Human CD45RA FITC L48

Human CD45RO PE UCHL-1

BD Multitest Human CD45RA/CD62L/CD3/CD4 Clone Quantity Number of Tests Cat. No.

Human CD3 PerCP SK7

20 μL/test 50 tests 340977Human CD4 APC SK3

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

50 tests 561438Human CD4 PerCP-Cy™5.5 SK3 5 μL/test

Human CD45RA FITC HI100 5 μL/test

Human CD197 (CCR7) Alexa Fluor® 647 150503 5 μL/test

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

9

BD Pharmingen Human Th1/Th2/Th17 Phenotyping Kit Clone Quantity Number of Tests Cat. No.

Human CD4 PerCP-Cy5.5 SK3

1 mL

50 tests 560751

Human IL-17A PE N49-653

Human IFN-γ FITC B27

Human IL-4 APC MP4-25D2

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

1 mL 50 tests 346048Human CD69 PE L78

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 25723.111 mL 50 tests 340456

Human IL-4 PE 3010.211

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.

Immunology: Intracellular Cytokines

BD T-cell cytokine kits, protocols, and software

templates for the BD Accuri C6 simplify the detection

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.

10

101 106.2102 103 104 105

101

106.

410

210

310

410

5FL

3 C

D8

PerC

P C

y5.5

-A

FL4 CD3 APC-A

C12 UnstimulatedGate: (Lymphocytes in all)

CD3+8+37.3%CD3+8+37.3%

101 106.2102 103 104 105

101

106.

210

210

310

410

5FL

2 C

D69

PE-

A

FL1 IFN gamma FITC-A

C12 UnstimulatedGate: (CD3+8+ in (Lymphocytes in all))

CD69+1.5%

CD69+IFN+0.1%

Q3-LL97.9%

IFN+0.5%

CD69+1.5%

CD69+IFN+0.1%

Q3-LL97.9%

IFN+0.5%

101 106.2102 103 104 105

101

106.

210

210

310

410

5FL

2 C

D69

PE-

A


D07 PMA+IonomycinGate: (CD3+8+ in (Lymphocytes in all))

CD69+27.4%

CD69+IFN+68.3%

Q3-LL1.0%

IFN+3.3%

CD69+27.4%

CD69+IFN+68.3%

Q3-LL1.0%

IFN+3.3%

A B C

BD Pharmingen Human Th1/Th2/Th17 Phenotyping Kit (Cat. No. 560751) analysis on the BD Accuri C6.Purified human PBMCs were stimulated with PMA/Ionomycin (at 50 ng/mL and 1 μg/mL, respectively) in the presence of BD GolgiStop 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 kit template, and analyzed using BD Accuri C6 software. Results: Density plots (gated on CD4+ lymphocytes) show that stimulated cells (right) were more likely than unstimulated controls (left) to produce high levels of IFN-γ, IL-4, and IL-17A as they differentiate into Th1, Th2, and Th17 helper T cells, respectively.

101 106102 103 104 105

101

106

102

103

104

105

FL2

IL-1

7A P

E-A

FL4 IL-4 APC-A

A05 UnstimulatedGate: (P1 in (R1 in all))

Q1-UL0.1%

Q1-UR0.0%

Q1-LL99.8%

Q1-LR0.1%

Q1-UL0.1%

Q1-UR0.0%

Q1-LL99.8%

Q1-LR0.1%

101 106102 103 104 105

101

106

102

103

104

105

FL2

IL-1

7A P

E-A

FL4 IL-4 APC-A

A06 PMA + IonomycinGate: (P1 in (R1 in all))

Q1-UL32.0%

Q1-UR0.3%

Q1-LL64.5%

Q1-LR3.2%

Q1-UL32.0%

Q1-UR0.3%

Q1-LL64.5%

Q1-LR3.2%

101 106102 103 104 105

102

106

103

104

105

FL1

IFN

gam

ma

FITC

-A

FL4 IL-4 APC-A

A05 UnstimulatedGate: (P1 in (R1 in all))

Q2-UL0.2%

Q2-UR0.0%

Q2-LL99.7%

Q2-LR0.1%

Q2-UL0.2%

Q2-UR0.0%

Q2-LL99.7%

Q2-LR0.1%

101 106102 103 104 105

102

106

103

104

105

FL1

IFN

gam

ma

FITC

-A

FL4 IL-4 APC-A

A06 PMA + IonomycinGate: (P1 in (R1 in all))

Q2-UL34.6%

Q2-UR0.4%

Q2-LL61.9%

Q2-LR3.1%

Q2-UL34.6%

Q2-UR0.4%

Q2-LL61.9%

Q2-LR3.1%

Unstimulated PMA+Ionomycin

101 107.2102 103 104 105 106

,500

500

01,

000

Co

un

t

FL4 CD3 APC-A

B04 1009 Unstim CocktailGate: Lymphocytes

CD3+77.4%CD3+77.4%

101 106.8102 103 104 105 106

101

105.

910

210

310

410

5FL

2 IL

4 PE

-A


B04 1009 Unstim CocktailGate: (CD3+ in Lymphocytes)

Q1-UL0.0%

Q1-UR0.0%

Q1-LL100.0%

Q1-LR0.0%

Q1-UL0.0%

Q1-UR0.0%

Q1-LL100.0%

Q1-LR0.0%

101 106.8102 103 104 105 106

101

105.

910

210

310

410

5FL

2 IL

4 PE

-A


B06 1009 Stim CocktailGate: (CD3+ in Lymphocytes)

Q1-UL0.4%

Q1-UR0.3%

Q1-LL67.4%

Q1-LR31.9%

Q1-UL0.4%

Q1-UR0.3%

Q1-LL67.4%

Q1-LR31.9%

A B C

Imm

un

olo

gy

Cytokine Production by Stimulated Naïve T CellsIFN-γ Production by Cytotoxic T Cells

IFN-γ and IL-4 Production by Activated T Cells

BD FastImmune IFN-γ/CD69/CD8/CD3 Kit (Cat. No. 346048) analysis on the BD Accuri C6.Human whole blood was drawn into heparinized tubes and stimulated with PMA (10 ng/mL) and Ionomycin (1 μg/mL) for 5 hours at 37°C in the presence of 10 ng/mL of Brefeldin A protein transport inhibitor (BD GolgiPlug™, Cat. No. 555029). Samples were harvested, fixed, lysed, permeabilized, and stained according to the kit procedure. 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+CD8+ cytotoxic T cells (A). Results: Compared to unstimulated controls (B), stimulated cells (C) were more likely to produce high levels of the activation marker CD69. Most of these CD69+ cells expressed IFN-γ as well. Data is representative of five donors.

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

BD Pharmingen FoxP3 Staining Kit Clone Quantity Number of Tests Cat. No.

Human FoxP3 Alexa Fluor® 647 259D/C7 1 vial

100 tests 560132

Human CD4 FITC RPA-T4 1 vial

Human CD25 PE M-A251 1 vial

Human FoxP3 Buffer A (10X) 25 mL


BD Pharmingen Regulatory T-Cell Cocktail Clone Quantity Number of Tests Cat. No.

Human CD4 FITC SK3

20 μL/test 50 tests 560249Human CD25 PE-Cy™7 2A3

Human CD127 Alexa Fluor® 647 hIL-7R-M21

BD Pharmingen Human Th17/Treg Phenotyping Kit Quantity Number of Tests Cat. No.

Human CD4 PerCP-Cy5.5

1 mL

50 tests 560762

Human IL-17 PE

Human FoxP3 Alexa Fluor® 647



BD GolgiStop Protein Transport Inhibitor (containing monensin) 0.7 mL

BD human Treg kits, protocols, and software templates

for the BD Accuri C6 simplify the detection and

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.

Immunology: Regulatory T Cells

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.


12

101.2 105.1102 103 104101.

510

5.3

102

103

104

FL2

IL-1

7 PE

-A

FL4 FoxP3 Alexa Fluor® 647-A

B09 UnstimulatedGate: (CD4+ in (Lymphocytes in all))

Th170.1%

Q7-UR0.0%

Q7-LL92.3%

Treg7.5%

Th170.1%

Q7-UR0.0%

Q7-LL92.3%

Treg7.5%

101.2 105.1102 103 104101.

510

5.3

102

103

104

FL2

IL-1

7 PE

-A


C03 PMA Ionomycin stimGate: (CD4+ in (Lymphocytes in all))

Th171.7%

Q7-UR0.2%

Q7-LL89.1%

Treg9.0%

Th171.7%

Q7-UR0.2%

Q7-LL89.1%

Treg9.0%

A B

100 105.9101 102 103 104 105

075

020

040

060

0C

ou

nt

FL2 FoxP3 PE-A

A06 Donor_1060 CocktailGate: (Non-Treg in (CD4 positives in Lymphocytes))

100 105.9101 102 103 104 105

7020

040

60C

ou

nt

FL2 FoxP3 PE-A

A06 Donor_1060 CocktailGate: (Tregs in (CD4 positives in Lymphocytes))

101 105.9102 103 104 105

101

105.

910

210

310

410

5FL

4 C

D12

7 A

lexa

Flu

or®

647

-A

FL3 CD25 PE-Cy7-A

A06 Donor_1060 CocktailGate: (CD4 positives in Lymphocytes)

Non-Treg75.9%

Tregs6.0%

Non-Treg75.9%

Tregs6.0%

A B C

102 105.2103 104

101

106.

210

210

310

410

5FL

2 C

D 2

5 PE

-A


A05 FoxP3 staining kieGate: (CD4 in (Lymphs in all))

Q7-UL18.0%

Q7-UR8.7%

Q7-LL71.9%

Q7-LR1.4%

Q7-UL18.0%

Q7-UR8.7%

Q7-LL71.9%

Q7-LR1.4%

102 106.2103 104 105

102

105.

210

310

4FL

4 Fo

xP3

Ale

xa F

luo

r® 6

47-A

FL1 CD4 FITC-A

A05 FoxP3 staining kitGate: (CD25 in (Lymphs in all))

Q6-UL0.6%

Q6-UR25.7%

Q6-LL21.1%

Q6-LR52.6%

Q6-UL0.6%

Q6-UR25.7%

Q6-LL21.1%

Q6-LR52.6%

A B

Identifying Tregs Using FoxP3

Distinguishing Th17 Cells and Tregs in a Single Sample

BD Pharmingen FoxP3 Staining Kit (Cat. No. 560132) analysis on the BD Accuri C6.Human PBMCs were stained with CD4 FITC and CD25 PE, fixed, permeabilized, and stained for intracellular content with FoxP3 Alexa Fluor® 647 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. Results: A. Gating on CD4+ lymphocytes, Tregs are CD25+FoxP3+. B. Gating on CD25+ lymphocytes, Tregs are CD4+FoxP3+. Both plots depict equivalent percentages of Tregs.

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.

Distinguishing Tregs Using CD127

BD Pharmingen Human Regulatory T-Cell Cocktail (Cat. No. 560249) analysis on the BD Accuri C6.Human PBMCs were stained according to the kit procedure. The cells were then fixed, lysed, and permeabilized using the BD Pharmingen™ Human Transcription Factor Buffer Set (Cat. No. 562574) and stained with PE-conjugated anti-human BD Pharmingen™ FoxP3 monoclonal antibody (Cat. No. 560082). 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: A. A CD25 vs CD127 plot was used to identify CD25brightCD127dim Tregs and non-Treg CD4+ cells. B, C. Tregs identified in Panel A were validated using FoxP3 staining. CD4+ cells identified as Tregs (C) expressed higher levels of FoxP3 than did non-Tregs (B).

Imm

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13

BD Stemflow Human Pluripotent Stem Cell Sorting and Analysis Kit

Clone Quantity Number of Tests Cat. No.

TRA-1-81 Alexa Fluor® 647 TRA-1-81 1.5 mL

50 tests 560461SSEA-1 FITC MC480 1.5 mL

SSEA-3 PE MC631 1.5 mL

Controls and compensation particles as detailed in kit manual

BD Stemflow Human Pluripotent Stem Cell Transcrip-tion Factor Analysis Kit


Nanog PE N31-355 1.5 mL

50 tests 560589Oct3/4 PerCP-Cy5.5 40/Oct-3 1.5 mL

Sox2 Alexa Fluor® 647 245610 1.5 mL


BD Stemflow Human and Mouse Pluripotent Stem Cell Analysis Kit


Oct3/4 PerCP-Cy5.5 40/Oct-3 1.5 mL

50 tests 560477SSEA-4 Alexa Fluor® 647 MC813 1.5 mL

SSEA-1 PE MC480 1.5 mL


BD pluripotent stem cell kits, protocols, and

software templates for the BD Accuri C6 simplify the

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.

Stem Cells: Pluripotent Stem Cells

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.


14

101 107.2102 103 104 105 106

101

107.

210

210

310

410

510

6FL

2 PE

SSE

A-3

-A

FL1 FITC SSEA-1-A

B03 KitGate: (P3 in all)

Q4-UL88.8%

Q4-UR2.7%

Q4-LL7.9%

Q4-LR0.6%

Q4-UL88.8%

Q4-UR2.7%

Q4-LL7.9%

Q4-LR0.6%

A

101 107.2102 103 104 105 106

101

107.

210

210

310

410

510

6FL

4 A

lexa

647

TR

A-1

-81-

A

FL1 FITC SSEA-1-A


Q5-UL81.6%

Q5-UR5.1%

Q5-LL12.2%

Q5-LR1.1%

Q5-UL81.6%

Q5-UR5.1%

Q5-LL12.2%

Q5-LR1.1%

B

101 107.2102 103 104 105 106

101

107.

210

210

310

410

510

6FL

4 A

lexa

647

TR

A-1

-81-

A

FL2 PE SSEA-3-A


Q6-UL5.2%

Q6-UR84.0%

Q6-LL1.7%

Q6-LR9.1%

Q6-UL5.2%

Q6-UR84.0%

Q6-LL1.7%

Q6-LR9.1%

C

101 107.2102 103 104 105 106

101

107.

210

210

310

410

510

6FL

2 PE

SSE

A-3

-A

FL1 FITC SSEA-1-A


Q4-UL88.8%

Q4-UR2.7%

Q4-LL7.9%

Q4-LR0.6%

Q4-UL88.8%

Q4-UR2.7%

Q4-LL7.9%

Q4-LR0.6%

A

101 107.2102 103 104 105 106

101

107.

210

210

310

410

510

6FL

4 A

lexa

647

TR

A-1

-81-

A

FL1 FITC SSEA-1-A


Q5-UL81.6%

Q5-UR5.1%

Q5-LL12.2%

Q5-LR1.1%

Q5-UL81.6%

Q5-UR5.1%

Q5-LL12.2%

Q5-LR1.1%

B

101 107.2102 103 104 105 106

101

107.

210

210

310

410

510

6FL

4 A

lexa

647

TR

A-1

-81-

A

FL2 PE SSEA-3-A


Q6-UL5.2%

Q6-UR84.0%

Q6-LL1.7%

Q6-LR9.1%

Q6-UL5.2%

Q6-UR84.0%

Q6-LL1.7%

Q6-LR9.1%

C

101 107.2102 103 104 105 106

101

107.

210

210

310

410

510

6FL

2 PE

SSE

A-3

-A

FL1 FITC SSEA-1-A


Q4-UL88.8%

Q4-UR2.7%

Q4-LL7.9%

Q4-LR0.6%

Q4-UL88.8%

Q4-UR2.7%

Q4-LL7.9%

Q4-LR0.6%

A

101 107.2102 103 104 105 106

101

107.

210

210

310

410

510

6FL

4 A

lexa

647

TR

A-1

-81-

A

FL1 FITC SSEA-1-A


Q5-UL81.6%

Q5-UR5.1%

Q5-LL12.2%

Q5-LR1.1%

Q5-UL81.6%

Q5-UR5.1%

Q5-LL12.2%

Q5-LR1.1%

B

101 107.2102 103 104 105 106

101

107.

210

210

310

410

510

6FL

4 A

lexa

647

TR

A-1

-81-

A

FL2 PE SSEA-3-A


Q6-UL5.2%

Q6-UR84.0%

Q6-LL1.7%

Q6-LR9.1%

Q6-UL5.2%

Q6-UR84.0%

Q6-LL1.7%

Q6-LR9.1%

C

102 106103 104 105102.

710

610

410

5FL

1 SS

EA-1

PE-

A

FL3 Oct3-4 PerCP-Cy5.5-A

D02 05_Ab stainedGate: (P1 in all)

Q1-UL0.2%

Q1-UR1.0%

Q1-LL27.5%

Q1-LR71.4%

Q1-UL0.2%

Q1-UR1.0%

Q1-LL27.5%

Q1-LR71.4%

102 107103 104 105 106102.

510

610

310

410

5FL

1 SS

EA-1

PE-

A

FL4 SSEA-4 Alexa 647-A


Q2-UL0.0%

Q2-UR1.5%

Q2-LL0.0%

Q2-LR98.5%

Q2-UL0.0%

Q2-UR1.5%

Q2-LL0.0%

Q2-LR98.5%

102 107103 104 105 106102.

710

610

410

5FL

3 O

ct3-

4 Pe

rCP-

Cy5

.5-A

FL4 SSEA-4 Alexa 647-A


Q3-UL0.0%

Q3-UR75.0%

Q3-LL0.0%

Q3-LR25.0%

Q3-UL0.0%

Q3-UR75.0%

Q3-LL0.0%

Q3-LR25.0%

A B C

102.7 106104 105102.

710

610

410

5FL

2 N

ano

g P

E-A

FL3 Oct3-4 PerCP-Cy5.5-A

F02 08_Ab stainedGate: (P1 in all)

Q8-UL0.6%

Q8-UR95.1%

Q8-LL2.6%

Q8-LR1.7%

Q8-UL0.6%

Q8-UR95.1%

Q8-LL2.6%

Q8-LR1.7%

102.6 106103 104 105102.

710

610

410

5FL

2 N

ano

g P

E-A

FL4 Sox2 Alexa 647-A


Q9-UL6.9%

Q9-UR88.7%

Q9-LL2.4%

Q9-LR2.0%

Q9-UL6.9%

Q9-UR88.7%

Q9-LL2.4%

Q9-LR2.0%

102.6 106103 104 105102.

710

610

410

5FL

3 O

ct3-

4 Pe

rCP-

Cy5

.5-A

FL4 Sox2 Alexa 647-A


Q10-UL7.0%

Q10-UR90.2%

Q10-LL2.3%

Q10-LR0.5%

Q10-UL7.0%

Q10-UR90.2%

Q10-LL2.3%

Q10-LR0.5%

A B C

Stem C

ells

Analyzing Stem Cells for Key Transcription Factors

Analyzing Stem Cells for Key Pluripotency Markers

Characterizing Stem Cell Pluripotency and DIfferentiation State

BD Stemflow Human Pluripotent Stem Cell Transcription Factor Analysis Kit (Cat. No. 560589) analysis on the BD Accuri C6.H9 human embryonic stem cells (hESCs) were disassociated using BD™ Accutase Cell Detachment Solution (Cat. No. 561527), stained according to kit instructions, and acquired on a BD Accuri C6 flow cytometer using the kit template. Cells were gated on light scatter properties of H9 hESCs and analyzed for expression of core pluripotency transcription factors using BD Accuri C6 software. Results: Most analyzed cells expressed the core pluripotency transcription factors Nanog, Oct3/4, and Sox2.

BD Stemflow Human Pluripotent Stem Cell Sorting and Analysis Kit (Cat. No. 560461) analysis on the BD Accuri C6.H9 hESCs were disassociated using BD Accutase Cell Detachment Solution (Cat. No. 561527), stained according to kit instructions, and acquired on a BD Accuri C6 flow cytometer using the kit template. Cells were gated on light scatter properties of H9 hESCs and analyzed for expression of key pluripotency surface markers using BD Accuri C6 software. Results: Most analyzed cells expressed positive pluripotency surface markers SSEA-3 and TRA-1-81, while few expressed the negative pluripotency marker (positive differentiation marker) SSEA-1.

BD Stemflow Human and Mouse Pluripotent Stem Cell Analysis Kit (Cat. No. 560477) analysis on the BD Accuri C6.H9 hESCs were disassociated using BD Accutase Cell Detachment Solution (Cat. No. 561527), stained according to kit instructions, and acquired on a BD Accuri C6 flow cytometer using the kit template. Cells were gated on light scatter properties of H9 hESCs and analyzed for expression of key pluripotency surface markers and transcription factors using BD Accuri C6 software. Results: Most analyzed cells expressed the positive pluripotency surface marker SSEA-4 and the pluripotency transcription factor Oct3/4, while few expressed the negative pluripotency marker (positive differentiation marker) SSEA-1.

15

A

B

C

D

E

F

Beads Wash

Detector Antibodies

Sample

NIR

Red

Analyze byFlow Cytometry

Concentration

PE MFI

MFI

Concentration (pg/mL)

Standard Curve

Bead-based flow cytometric immunoassays are

powerful methods of quantifying proteins because

they allow researchers to multiplex many analytes

with very little sample.

Bead-Based Assays: BD Cytometric Bead Array Kits

There are two ways to use BD CBA: preconfigured kits (described here) and fully configurable flex sets (described further at bdbiosciences.com/go/cba/).

BD CBA kits provide preconfigured panels of 3–7 analytes for ultimate ease of use. For example, the BD™ CBA Human Th1/Th2/Th7 Cytokine Kit can quantitate IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A simultaneously. Other kits include panels of human cytokines, chemokines, and anaphylatoxins; mouse cytokines and immunoglobulins; and non-human primate cytokines.

On the BD Accuri C6, the beads are excited by the red laser and detected in FL4, while the PE reporter is excited by the blue laser and detected in FL2. A BD Accuri C6 software template simplifies setup and acquisition. Results can be displayed in FCAP Array™ software by bead (graphing one analyte across all samples) or by sample (graphing one sample across all analytes).

BD CBA Kit Bead Resolution

BD CBA kit bead resolution on the BD Accuri C6.The beads used in BD CBA kits are resolved clearly in the FL4 detector of the BD Accuri C6 using the standard 675/25 filter.

103 107.2104 105 106

020

050

100

150

Co

un

t

FL4 675/25

104 106.8105 106103.

610

6.1

104

105

FL3

780/

60

FL4 675/25

16

Brand Kit Cat. No.

BD™ Cytometric Bead Array Human Anaphylatoxin Kit 561418

BD™ Cytometric Bead Array Human Chemokine Kit 552990

BD™ Cytometric Bead Array Human Inflammatory Cytokine Kit 551811

BD™ Cytometric Bead Array Human Inflammatory Cytokine Lyophilized Standard 552932

BD™ Cytometric Bead Array Human Th1/Th2 Cytokine Kit II 551809

BD™ Cytometric Bead Array Human Th1/Th2 Cytokine Kit 550749

BD™ Cytometric Bead Array Human Th1/Th2 Cytokine Lyophilized Standard 551810

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.

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

BD CBA kit analysis by analyte or sample.Human PBMCs were cultured for several days with plate-bound anti-CD3, soluble anti-CD27, IL-2, and IL-4. Cells were stimulated with PMA+Ionomycin for several hours before collecting culture supernatants. Samples were prepared and stained with the BD CBA Human Th1/Th2/Th17 Cytokine Kit (Cat. No. 560484), acquired on a BD Accuri C6 using the BD CBA Kit Accuri template, and analyzed using FCAP Array software v3.0.1. A. Bar chart of human IFN-γ levels for all standards and test samples. B. Bar chart of all seven cytokine levels for one sample.

Bead

-Based

Assays

A B

17

102

103

103 103 103

104

105

Green fluorescence (533 nm)

Red

flu

ore

scen

ce (

670

nm

) LNA: low nucleic acid content cellsHNA: high nucleic acid content cells

background

bacteria

HNA

LNA 102

103

103 103 103

104

105

102

103

104

105


Red

flu

ore

scen

ce (

670

nm

) damaged cells

intact cells

103 104 105


Bac

teri

a ce

ll co

un

t

Water sample 1Water sample 2Water sample 3

A B C

The template and product information sheet are available on the BD Biosciences website.

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 BD Accuri™ C6 Eawag Water Quality Template2

simplifies the enumeration of intact and damaged

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.

Microbiology: Water Quality

Using nucleic acid dyes, flow cytometry can quantitate microbes and discriminate them from debris much more rapidly than plate methods. SYBR® Green I, which preferentially stains double-stranded DNA, is well suited for staining bacteria in water samples for flow cytometric analysis. PI can be added to differentiate live and damaged bacterial cells. PI is impermeable to healthy cells with intact membranes, but permeates cells with compromised membranes such as dead cells.

For a detailed discussion of the use of the Eawag template and protocol for water quality research, see the BD Biosciences white paper, Assessing Water Quality with

the BD Accuri™ C6 Flow Cytometer (January 2013), available at bdbiosciences.com.

2. The water quality template and staining protocol were developed in collaboration with Eawag, the Swiss Federal Institute of Aquatic Science and Technology.

Enumerating Intact and Damaged Bacteria in Drinking Water

Water quality analysis using the Eawag water quality template on the BD Accuri C6.Drinking water samples were stained according to the Eawag protocol, acquired on a BD Accuri C6 using the Eawag water quality template, and analyzed using BD Accuri C6 software. A. When a sample is stained with SYBR® Green I, all bacteria appear within the template’s single, fixed gate, while noise and debris are excluded. B. When the sample is co-stained with SYBR® Green I and 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.

Mic

rob

iolo

gy

18

Co

un

ts

FL1-H

02,

000

4,00

06,

400

101 102 103 104 105 106 107.2

Co

un

ts

FL2-H

02,

000

4,00

06,

400

101 102 103 104 105 106 107.2

Co

un

ts

FL3-H

02,

000

4,00

06,

400

101 102 103 104 105 106 107.2

Co

un

ts

FL4-H

02,

000

4,00

06,

400

101 102 103 104 105 106 107.2

Co

un

ts

FL1-H

02,

000

4,00

06,

400

101 102 103 104 105 106 107.2

Co

un

ts

FL2-H

02,

000

4,00

06,

400

101 102 103 104 105 106 107.2

Co

un

ts

FL3-H

02,

000

4,00

06,

400

101 102 103 104 105 106 107.2

Co

un

ts

FL4-H

02,

000

4,00

06,

400

101 102 103 104 105 106 107.2

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

BD Accuri™ Spherotech Validation Beads contain a

mixture of fluorochromes that are spectrally similar to

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.

Instrument Setup: Validation Beads

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.

BD Accuri™ Spherotech 6-peak Validation Beads (Cat. No. 653145) are used to validate performance of the FL4 (APC) detector.

For detailed procedural and troubleshooting steps to use in validating the BD Accuri C6, see Chapter 2 of the BD Accuri™ C6 Software User Guide, available at bdbiosciences.com.

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

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.

Validating Performance of the BD Accuri C6

Instru

men

t Setup

19

23-16217-00

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.

Alexa Fluor® and SYBR are registered trademarks of Life Technologies Corporation.

FCAP Array is a trademark of Soft Flow Hungary Ltd.

Microsoft, Windows, and Windows Vista are registered trademarks of Microsoft Corporation.

Spherotech is a trademark of Spherotech, Inc.

BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2014 BD

BD Biosciencesbdbiosciences.com

BD Biosciences Regional Offices

Office locations are available on our websites.

AustraliaToll Free 1800.656.100Tel 61.2.8875.7000Fax 61.2.8875.7200bdbiosciences.com/anz

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