The BD Pharmingen™ Apoptosis, DNA Damage and

BD Apoptosis, DNA Damage and Cell Proliferation
Kit and Template
Cell Status Assays on the BD Accuri™ C6 Flow Cytometer
Features
Preconfigured kit, protocol, and software template
Assess apoptosis, DNA damage, and cell proliferation on
the BD Accuri C6
Support studies involving cleaved PARP, H2AX, and BrdU
Conserve precious sample by measuring multiple
parameters in a single tube
Enable quick and easy setup and analysis using the
BD Accuri C6
The BD Pharmingen™ Apoptosis, DNA Damage and
Cell Proliferation Kit (Cat. No. 562253), protocol, and
software template for the BD Accuri™ C6 flow cytometer
simplify the simultaneous analysis of important cellular states
in human samples. The kit, which supports studies using
bromodeoxyuridine (BrdU), phosphorylated H2AX, and cleaved
Poly (ADP-ribose) polymerase-1 (PARP), includes fluorescent
antibodies, buffer systems, and other reagents needed for
acquisition and analysis. A BD Accuri™ C6 software template
matched to the kit includes a predefined workspace, markers,
regions, gates, and parameter names for quick and easy setup
and analysis.
0
2,000,000
FSC-A
2,919,281
10 6.1
10 5
10 4
10 3
10 2
FL4 H2AX pS139 Alexa Fluor® 647-A
1,000,000
Q5-UR
34.3%
Q5-LL
40.9%
10 3
Q5-LR
12.3%
10 4
10 5
10 6.4
To use the kit, incubate the cells with BrdU (included) to
incorporate it into proliferating cells. Then, stain the cells for cell
surface markers, if desired. Fix and permeabilize the samples and
treat them with DNase, which helps to expose the BrdU epitopes.
Stain the cells simultaneously with fluorochrome-labeled antiBrdU, cleaved PARP, and H2AX. Finally, resuspend the cells in
staining buffer and analyze them on the BD Accuri C6 using the
free software template.
B06 Aphidicolin
Gate: (Cells in all)
Q5-UL
50.7%
Q5-UR
1.7%
10 5
10 6.1
FL3 BrdU PerCP-Cy5.5-A
FL4 H2AX pS139 Alexa Fluor® 647-A
Cells
93.9%
Q5-UL
12.4%
10 4
FSC-A
B05 DMSO
Gate: (Cells in all)
10 2.2
2,919,281
Many factors, including stress, radiation, environmental exposure,
and treatment with small molecules, can lead to changes in
apoptosis, DNA damage, cell proliferation, and other cellular
events. The kit uses three antibodies to detect these events. It
identifies proliferating cells with antibodies to BrdU, which has
previously been incorporated into the cells’ newly synthesized
DNA. It identifies damaged DNA with antibodies to H2AX, which
is rapidly phosphorylated at DNA strand breaks (and less rapidly
at replicating DNA sites). Finally, the kit identifies apoptotic
cells with antibodies to a PARP fragment, cleaved from PARP by
caspase-3 in the early phases of apoptosis.
10 3
2,000,000
B06 Aphidicolin
Gate: [No Gating]
500,000
SSC-A
1,000,000
0
Aphidicolin
994,570
0
10 2
Cells
93.2%
500,000
DMSO
B05 DMSO
Gate: [No Gating]
0
SSC-A
994,570
Figures 1 and 2 show data on the BD Accuri C6 using the
preconfigured kit and software template.
Q5-LL
47.6%
10 2.2
10 3
Q5-LR
0.0%
10 4
10 5
10 6.4
FL3 BrdU PerCP-Cy5.5-A
Figure 1. Analysis of cell proliferation on the BD Accuri C6
Jurkat cells (human T-cell leukemia: ATCC TIB-152) were treated with compound
vehicle (DMSO) or the S-phase inhibitor aphidicolin (3 µg/mL) for 4 hours at 37°C.
BrdU (10 µM) was added from the BD Pharmingen Apoptosis, DNA Damage and
Cell Proliferation Kit (Cat. No. 562253) during the last hour of treatment. The
cells were harvested and stained according to the kit instructions, acquired on a
BD Accuri C6 using the kit template, and analyzed using BD Accuri C6 software.
Results: A light scatter gate (Cells) was drawn around the cell population (left
plots). With DMSO treatment alone (upper plots), 46.6% of the cells were
proliferating (BrdU+, UR and LR). After treatment with aphidicolin (lower plots),
proliferating cells (BrdU+, UR and LR) all but vanished, while the percentage and
signal intensity of H2AX expression remained similar to the control.
Visit bdbiosciences.com for more information.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Easy to use, simple to maintain, and affordable, the BD Accuri
C6 personal flow cytometer (Cat. No. 653118) is equipped with
a blue laser, a red laser, two light scatter detectors, and four
fluorescence detectors. A compact design, fixed alignment,
and pre-optimized detector settings result in a system that is
simple to use. A nonpressurized fluidics system enables kinetic
measurements in real time. For walkaway convenience, the
optional BD CSampler™ accessory (Cat. No. 653124) offers
automated sampling from 24-tube racks or multiwell plates.
BD Apoptosis, DNA Damage and Cell Proliferation Kit and Template
0
10 3
10 4
10 5
10 7.1
Q2-UR
0.6%
Q2-LL
52
52.1%
.1%
Q2-LR
1
1.2%
.2%
10 4
10 5
10 6
Q2-UL
46.1%
10 3
FL4 H2AX pS139 Alexa Fluor® 647-A
10 5.9
10 2
10 6.4
10 4
10 5
Q1-LR
1.8%
10 3
FL3 BrdU PerCP-Cy5.5-A
2,919,281
Q1-LL
51
51.4%
.4%
10 1.6
10 5.9
10 4
10 5
10 5.9
Q2-UL
73.7%
Q2-UR
19.8%
Q2-LL
5.5%
Q2-LR
1.0%
10 6
10 7.1
B04 Camptothecin
Gate: (Cells in all)
10 5
Q1-LR
17.0%
10 4
10 5
10 4
Q1-LL
73.3%
10 3
10 4
10 3
Q1-UR
3.9%
10 2
Q1-UL
5.8%
FL4 H2AX pS139 Alexa Fluor® 647-A
F
B04 Camptothecin
Gate: (Cells in all)
10 1.6
10 3
FL2 Cleaved PARP PE-A
10 5
10 6.4
2,000,000
FSC-A
Q1-UR
0.1%
10 3
FL3 BrdU PerCP-Cy5.5-A
1,000,000
Q1-UL
46.7%
B05 DMSO
Gate: (Cells in all)
FL2 Cleaved PARP PE-A
E
Cells
86.9%
C
B05 DMSO
Gate: (Cells in all)
10 1.6
2,919,281
B04 Camptothecin
Gate: [No Gating]
500,000
994,570
2,000,000
FSC-A
0
SSC-A
Camptothecin
D
1,000,000
10 2.2
0
10 2.2
Cells
93.2%
500,000
994,570
B
B05 DMSO
Gate: [No Gating]
0
SSC-A
DMSO
A
10 1.6
FL2 Cleaved PARP PE-A
10 3
10 4
10 5
10 5.9
FL2 Cleaved PARP PE-A
Figure 2. Analysis of apoptosis, DNA damage, and cell proliferation on the BD Accuri C6
Jurkat cells (human T-cell leukemia: ATCC TIB-152) were treated with compound vehicle (DMSO) or the topoisomerase I inhibitor camptothecin (6 µM) for 4 hours at
37°C to induce apoptosis. BrdU (10 µM) was added from the BD Pharmingen Apoptosis, DNA Damage and Cell Proliferation Kit (Cat. No. 562253) during the last hour
of treatment. The cells were harvested and stained according to the kit instructions, acquired on a BD Accuri C6 using the kit template, and analyzed using BD Accuri C6
software. Results: With DMSO treatment alone (upper plots), approximately 46.7% of the cells were proliferating (BrdU+, plot B) and very few were apoptotic (cleaved
PARP+, plots B and C). The DMSO control had a low signal intensity of H2AX expression with 46.1% of the non-apoptotic cells staining positive (plot C). After treatment
with camptothecin (lower plots), apoptotic cells (cleaved PARP+, plots E and F) increased and proliferating cells (BrdU+, plot E) decreased, as expected. Among non-apoptotic
cells, H2AX expression increased both in percentage (73.7%) and signal intensity (plot F), indicating DNA damage.
Ordering Information
All kits and their associated software templates are available at bdbiosciences.com/go/templates.
Description
Clone
Quantity
Number of Tests
Cat. No.
BD Pharmingen™ Apoptosis, DNA Damage and Cell Proliferation Kit, containing:
BrdU PerCP-Cy™5.5
3D4
250 µL
H2AX (pSer139) Alexa Fluor® 647
N1-431
250 µL
Cleaved PARP (Asp214) PE
F21-852
250 µL
BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution
25 mL
BD Perm/Wash™ Buffer
25 mL
BD Cytofix/Cytoperm™ Plus Permeabilization Buffer
10 mL
DAPI (not used with BD Accuri C6)
100 µL
BrdU solution
0.5 mL
DNAse
300 µL
50 tests
562253
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Class 1 Laser Product.
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