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Cell Proliferation and Apoptosis: Two Sides of a Coin
Monisha Sundarrajan, PhDSenior Scientist
Research Applications Support
For Research Use Only. Not for use in diagnostic or therapeutic
procedures.
3
Cell Proliferation
Cell proliferation is defined as an increase in the number of cells as a result of cell growth and division.
Uncontrolled cell growth or proliferation is the hallmark of cancer cells.
4
The Cell Cycle and its Phases
MM
GG11
SS
GG00
cdk2
cyclin E
Cdk
4,6
cyclin D
cdc2
cyclin A,B
p27
p53
p21
GG22
5
Cell Proliferation Application Decision Tree
Cell proliferation
Bioimaging/IHC
BrdU incorporation
Ki-67, p-Histone H3,cyclins, etc
CBA Phosphorylations as indicators of cell proliferation/cytokine production
Live
BD Horizon™Violet Proliferation Dye 450
7-AAD and PI
Fixed
BrdU incorporation
Ki-67, p-Histone H3, PCNA, cyclins, RB, cytokines
Western blot/IP
Cell cycle markersp-Histone H3, PCNA
Fixed
BrdU incorporation, Ki-67, p-Histone, cyclins, etc
Intact cells Cell extracts Tissue sections
Treg suppression assay/functional assay suppression of effector cells assessed by CD25/CD69 expression
Phosphorylations as indicators of cell proliferation
Bioimaging/microscopy
Flow cytometry
6VPD 450
Day 1
Day 2
Day 3
Cell Proliferation Assessment Using Violet Proliferation Dye 450 (VPD 450)
Experimental design:
Enrich mouse spleen by positive selection via CD4+
enrichment.
Load isolated cells with VPD 450, 1 μM, for 10 minutes.
Harvest CD3/CD28 stimulated cells on the days indicated.
Analyze by flow cytometry.
7
Day 1
Day 2
Day 3
Condition: CD3/CD28
VPD 450
IL-2
Alexa
Fluor
®48
8
Experimental design:
Enrich Balb/c spleen by positive selection via CD4+
enrichment.
Load isolated cells with VPD 450, 1 μM for 10 minutes.
Stimulate cells with soluble antiwith soluble anti--
CD3/CD28 (1ug) in the presence of transport inhibitor.
Fix/perm stain cells.
Simultaneous Assessment of Cell Proliferation and IL-2 Secreted by Cells During T-cell Stimulation
8
Cell Cycle Analysis of Population Stained for Incorporated BrdU and Total DNA Levels (7-AAD)
Human PBMCs were stimulated with anti-CD3/CD28 for 48 hours and re-stimulated with PMA+Ionomycin for 4 hours, and BrdU was added for the final 1 hour. Cells were then harvested and stained using the BrdU staining protocol.
BrdU
-FIT
C
BrdU
-APC
Sub-G0/G1
S phaseS phase
G2+M phase
G0/G1
Sub-G0/G1
G2+M phase
G0/G1
9
Stimulated Splenocytes Assessed for Cell Proliferation Using VPD 450 and anti-BrdU Ab Simultaneously
Experimental design:
Mouse splenocytes were incubated with 1 μMVPD 450 for 10 minutes and stimulated with anti-CD3/CD28 for 48 hours. Cells were pulsedwith BrdU for 1 hour, prior to harvest.
Cells were harvested and stained using theBrdU
staining protocol and analyzed by flowcytometry.
VPD 450
VPD 450
BrdU
-APC
Condition: CD3/CD28 Stimulation for 48 hours
10
Ki-67: Another Marker for Cell Proliferation
Ki-67 is expressed in Go/G1 (P5 gated cells) and post mitotic G2/M phase (P4 gated) cells (data generated at BD Biosciences).
Ki-67 Alexa Fluor®
700
BrdU
Per
CP-C
y™5.
5
Ki-6
7 A
lexa
Flu
or®
700
DAPI-ADAPI-ASSC-A DAPI-A
BrdU
Per
CP-C
y5.5
FSC-
A
DA
PI-
W
G2/MG0/G1
S phase
11
Cell Cycle Analysis on HeLa Cells Treated with Aphidicolin (DNA Topisomerase α
Inhibitor) Monitored by BrdU
Staining
No treatment 1 μg Aphidicolin
BrdU
0
10
20
30
40
50
60
1.00E-09 1.00E-08 1.00E-07 1.00E-06 1.00E-05
Dose (on log scale)
% B
rdU
pos
itive EC50:
1.92E-07
The images were captured on a BD Pathway™
855 bioimaging system with a 20x objective and merged using BD Attovision™
software.
Hoechst
-
blueBrdU
-
redHistone
-
yellowTubulin
-
green
12
Cell Proliferation Assessed in Mouse Small Intestinal Sections by BrdU Staining
The images were captured on a BD Pathway™
435 bioimaging system with a 20x objective and merged using BD Attovision software.
Actin
-
greenβ-Tubulin-
redBrdU
–
yellowHoechst-
blue
13
•
Characteristics of Tregs:
proliferate very slowly
•
Hallmark assays to assess Treg functionsSuppress proliferation in effector cellsSuppress cytokine production by effector cells
•
Salient markers for Tregs–
Surface: CD4, CD25, CD127–
Intracellular: FoxP3
Regulatory T Cells (Tregs)
Regulatory T cells, also called “Tregs,”
play an important role in maintaining immunological unresponsiveness
to self antigens (self tolerance) and control of immune responses to foreign antigens.
14
Experimental design
Mouse splenocytes were stimulated with CD3/CD28
aand cultured
for 5 days. At the end of 5 days, cells were further cultured and then re-stimulated for 5 hours with PMA+Ionomycin. The cells were incubated with 100 μM BrdU for the final hour of culture and then harvested.
Following harvesting, the cells were stained with anti-CD4 PerCP-Cy5.5 and Foxp3 Alexa Fluor®
647 using the Foxp3 staining protocol.
Upon completion of FoxP3 staining, the cells were refixed and permeabilized
using BD Cytofix/Cytoperm™
buffer and stained with anti-BrdU Ab, allowing the detection of incorporated BrdU.
Staining of anti-BrdU with Foxp3 for Detection of Cell Proliferation in Mouse Tregs
BrdU FITC
Foxp
3–A
lexa
Flu
or®
647
2.3%
CD4 PerCP-Cy5.5
Foxp
3–A
lexa
Flu
or®
647
2.4% 0.4%
10.3%
15VPD
450
Foxp
3 Alexa
Fluor
®64
7
Day 1
Day 2
Day 3
Significant Insights into the Mechanism of Treg Proliferation as Assessed by Violet Proliferation
Dye 450 (VPD 450)
Experimental design
Enrich mouse splenocytes by positive selection via CD4+
enrichment.
Load isolated cells with VPD 450, 1 μM, for 10 minutes. CD3/CD28 stimulated cells were harvested on days as indicated
Fix and permeabilize cells using the Foxp3 staining protocol.
Condition: CD3/CD28
16
Expression of Ki-67 in Human Tregs
CD4+FoxP3+
2.9%
0.9%5.7% Ki-67+FoxP3+
Human PBMCs were stained for Ki-67 and FoxP3 using the FoxP3 staining protocol.
SSC
FoxP
3 PE
FSC
Ki-67 Alexa Fluor®
488
CD4 PerCP-Cy5.5
Ki-67-FoxP3+
CD4 PerCP-Cy5.5
FoxP
3 PE
SSC
17
Treg Suppression Assay Kit:Treg Suppression Assay Kit:
how the assay works
•
Tregs are sorted using CD4+, CD25+, CD127low/dim
and CD45RA+.
•
Cells are expanded in culture for 13 days.
•
Expanded Tregs are placed with effector cells (autologous PBMCs)
in the presence of T-cell specific stimulus (SEB, CD3/CD28, CD2/CD2R).
•
After 7 hours, the frequency of CD69-positive and/or CD154-positive effector T cells (response) is measured in the presence and absence of Tregs.
•
CD25 is used to identify and exclude Tregs during analysis.
•
The percent suppression of the response is calculated.
New Assay to Assess Treg Function: Suppression of Effector Cells
18
Treg-mediated Suppression of CD154 and CD69 in PBMCs Stimulated with CD3/CD28
CD4- CD4+
Tregs CD154
CD69
CD25
14.4%
34.1%MFI=6693
MFI=2597
CD154
CD69
22.6%
42.7%MFI=8943
MFI=3704
Cultured without Tregs Cultured with Tregs
19
Apoptosis
Definition:
The process leading to controlled self- destruction of a cell.
Cells undergo death neatly
without damaging their neighbors. Apoptosis is a “programmed event.”
20
Importance of Apoptosis•
Development–
Organs, appendages, patterning–
Thymic selection (lymphocyte development)•
Tissue homeostasis–
Tumor •
Cell termination–
Viral infection, cancer
21
Apoptotic Signaling Pathways
Fas
Procaspase 3
Caspase 8
Caspase 3
Bid
tBid
FADD
Procaspase 8
Bax/Bak
Procaspase 9Caspase 9
Procaspase 3
Caspase 3
Cytochrome c
Growth factor withdrawalIrradiationLoss of matrix contactGlucocorticoids
?
Bcl-2Bcl-X
DNA fragmentation
iCAD
CAD
Apaf-1↓Ψm
EXTRINSIC PATHWAY INTRINSIC PATHWAY
C-FLIPIAP’s
Smac
bdbiosciences.com/pathways
22
Hallmarks of Apoptosis• Plasma membrane alterations• Mitochondrial changes• Activation of caspases• DNA fragmentation
23
Apoptosis Application Decision TreeApoptotic cells
IHCActiveCaspase-3
Cleaved PARP
ELISA
ActiveCaspase-3
Cleaved PARP
Bioimaging/microscopyFlow cytometry
SpectrofluorometryCaspase activity
Live
Loss of membrane asymmetry-Annexin
V
Mitochondrial membrane potential-BD™
Mitoscreen
Fixed
Cleaved markers-Active Caspase3-Cleaved PARP
Caspase inhibitors-FAM-VAD-FMK
TUNEL/DNAfragmentation-APO-Direct-APO-BrdU
Western blot/IPApoptosis markers
Live
Annexin V
Fixed
Active Caspase-3Cleaved PARP
Caspase inhibitors-FAM-VAD-FMK
Intact cells Cell extracts Tissue sections
Caspase inhibitors-FAM-VAD-FMK
24
Apoptosis: Scatter Properties
Formation of apoptotic vesicles •Increases side scatter
Reduced refractive index of apoptotic cells •Decreases forward scatter
Cell shrinkage during apoptosis is associated with a decrease in
forward scatter. Analysis of light scatter is often combined with other assays.
Untreated Camptothecin treated
25
Annexin VAnnexin V is a surface marker and detects early membrane changes
associated with apoptosis.Pros:
Rapid confirmation of apoptosisUses live, unfixed cells
Applications•Flow cytometry (cells in suspension)•Fluorescence microscopy (adherent cells)
26
Detection of Membrane Changes by Annexin V Staining and Analysis by Flow Cytometry
V450-Annexin V
Rel
ativ
e C
ell N
umbe
r
Jurkat T cells were treated with 6 μM camptothecin for 4 hours. Cells were incubated with BD Horizon™
V450 Annexin V and analyzed by flow cytometry.
Untreated Camptothecin treated
V450-Annexin V
27
Detection of Changes in Mitochondrial Membrane Potential (JC-1)
JC-1: lipophilic cationic dye fluorescence is detected on a flow cytometer
J-aggregates (healthy cell)
monomers (apoptosis indicator)
J-aggregates (healthy cell)
monomers (apoptosis indicator)
28
Detection of Active Caspase 3: “Executioner”
of Apoptosis and its By-product: Cleaved PARP
TreatedTreatedUntreated Untreated
Jurkat T cells were treated with camptothecin, fixed and permeabilized with BD Cytofix/Cytoperm
buffer, and subsequently stained for active caspase 3 using anti-caspase Ab or cleaved PARP.
Untreated/control
Camptothecin treated (6 μM)
Caspase-3
Cleaved-PARP
29
Cleaved PARP Expression in Formalin-fixed, Paraffin-embedded Rat Lymph Node
Rat lymph nodes were stained with monoclonal cleaved PARP specific Ab F21-
852 using the biotin, streptavidin three-step detection method.
30
Detection of DNA Fragmentation During Apoptosis by “End Labeling”
or “TUNEL”
Using
the APO-DIRECT™
Kit
NonNon--
apoptotic apoptotic cellscells
NonNon--
apoptotic apoptotic cellscells
Positive apoptotic cells in SPositive apoptotic cells in S--
phasephase
31
Significant Tool in Drug Discovery Research: Assessing Cell Proliferation, DNA Damage, and
Apoptosis Using Flow Cytometry
`̀
3% 24%
14%59%
12% 31%
6%51%
Phos
H2A
X-A
PC
Untreated
15% 6%
19%60%
3% 1%
64%32%
Clea
ved
PARP
–APC
BrdU Alexa Fluor®
488
Camptothecin treatment, 5 μM
Experimental design
HeLa cells were untreated or treated with camptothecin 5 μM and BrdU for 4 hours.
Cells were then harvested and stained with anti-BrdU, H2AX, and cleaved PARP using the BrdU staining protocol.
32
Increase in Camptothecin Concentration and Incubation Time Leads to Increased H2AX and PARP Expression
and Loss of BrdU Incorporation
Phos
H2A
X A
PC
Clea
ved
PARP
APC
BrdU Alexa Fluor®
488
Camptothecin treatment 20 μM Experimental design
HeLa cells were untreated or treated with camptothecin,
20 μM, for 24 hours, further incubated for 48 hours post washing, and pulsed with BrdU for the final 1 hour.
Cells were then harvested and stained with anti-BrdU, H2AX, and cleaved PARP using the BrdU staining protocol.
32%0%
0%68%
49%
BrdU Alexa Fluor®
488
33
Acknowledgments
Jeanne Elia Jurg Rohrer Joyce Ruitenberg
Smita Ghanekar Christopher Boyce
Ravi Hingorani Cynthia Lane
Natalie Golts Martha Wilkinson
and others.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.Alexa Fluor® is a registered trademark of Molecular Probes, Inc.Cy™ is a trademark of Amersham Biosciences Corp.BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2010 BD23-12232-00