Apoptosis: Methods and Assay Selection Strategies

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Apoptosis Methods and Assay Selection Strategies

• 
  
  
  
  
  Presented by Lisa Stein, Ph.D for BD Biosciences
  Pharmingen

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Life and Death of the Cell
External Factors
Internal Factors
Status Quo
Divide
Die
Differentiate
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A Classification of Apoptosis Detection Methods

• Type of Change
• Cellular
• Nuclear
• Cell Surface
• Signaling proteins
• Mitochondria

Detection Method Microscopy, Flow cytometry Vital
dyes, TUNEL Annexin V Caspase pathway Mitochondria
l dyes (JC-1)
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Apoptosis Biology and Techniques
Techniques Cells Cell extracts Tissue
sections

• Biology
• Membrane changes
• Annexin V
• DNA fragmentation
• APO-BRDU/DIRECTTM
• Apoptosis signaling
• Caspases
• Active Caspases
• Caspase substrates
• Caspase inhibitors
• Mitochondria

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The Cell BiologyApoptosis Times
Circa 1995
Country Final 35
HUMAN GENES SAME AS WORMS Joe C. Elegans says he
is human. He has human genes. He was raised by a
pack of humans. As new Apoptosis reagents are
developed, worms are getting as smart as
humans. My genes are human, says Elegans,
continued on page 5...
InsideApoptosis
New Discovery
Genes bcl-2 caspase-3 Granzyme TRAIL FASL C
elegans Homology Receptor mediated
Whirlwind RIP MADD Fast K Requiem XIAP Bak Bax ba
d
I am human
Apo Inhibitor Found Cell death takes a holiday.
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Selecting and Analyzing Apoptosis Assays

• Measuring apoptosis through caspase activation
  and other mechanisms
• Biology and techniques
• Measuring caspase inhibition
• Inhibitors as tools to protect cells from
  apoptosis/promote cell survival
• Mitochondrial changes during caspase inhibition
• Inhibitors as tools to dissect apoptotic pathways
• Emerging apoptosis technology CBA

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Apoptosis Signaling
FasL
Receptor
Mitochondrial
Camptothecin
Staurosporine
Fas
FADD
DNA damage
?
Pro-caspase8
ZVAD
Active caspase8
Active Bid
Mitochondria
Pro-caspase9 Apaf-1 Cyt c
ZVAD
Active caspase9
Pro-caspase3 (6, 7)
ZVAD
Active caspase3 (6, 7)
DEATH
Massive substrate cleavage
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Apoptosis Decision Tree
Apoptotic Sample
Cells
Cell Extracts
Tissue Sections
Flow
Flow
Microscopy
ELISA
IHC
Western Blot / Immunoprecipitation
Spectrofluorometry
Gene Expression
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Changes in Light-Scatter during Apoptosis
Spontaneous Mouse Thymocyte Apoptosis
0 hr
24 hr
48 hr
SSC-H
FSC-H
Cell shrinkage during apoptosis is associated
with a decrease in forward scatter. Analysis of
light scatter is often combined with other assays.
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Apoptosis Signaling
FasL
Receptor
Mitochondrial
Camptothecin
Staurosporine
Fas
FADD
DNA damage
?
Pro-caspase8
ZVAD
Active caspase8
Active Bid
Mitochondria
Pro-caspase9 Apaf-1 Cyt c
ZVAD
Active caspase9
Pro-caspase3 (6, 7)
ZVAD
Active caspase3 (6, 7)
DEATH
Massive substrate cleavage
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Apoptotic Sample
Cells
Flow
Fixed
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Classical Technique Annexin Assay
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Annexin V Assay A Time Course
Jurkat T Cells
PI
Annexin V-FITC
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Caspase Activation
Mitochondrial
General
Proximity
p20
Pro
p10
Pro-caspase8
D
D
Pro-caspase


Apaf-1
FADD

Active Caspase8
Active Caspase
Active Caspase9 (apoptosome)
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Comparison of Annexin V and Active Caspase-3 in
Jurkat T Cells
Incubation with Camptothecin (hr)
Relative Cell Number
0 0.5 1
2 4
Annexin VPE
Active Caspase-3FITC
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Apoptosis Signaling
FasL
Receptor
Mitochondrial
Camptothecin
Staurosporine
Fas
FADD
DNA damage
?
Pro-caspase8
ZVAD
Active caspase8
Active Bid
Mitochondria
Pro-caspase9 Apaf-1 Cyt c
ZVAD
Active caspase9
Pro-caspase3 (6, 7)
ZVAD
Active caspase3 (6, 7)
DEATH
Massive substrate cleavage
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PARP is 116 kDa Nuclear Enzymeand Caspase
Substrate
Specificity of PARP Antibodies
HL60 cells Camptothecin (0-6 h time course)
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Caspase and Caspase Substrates
Fas-induced Apoptosis Jurkat T Cells
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Caspase Executionior and Substrate
Active Caspase-3
Cleaved PARP
1
Control
1
Cell Count
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Camptothecin4 µM, 4 h
40
PE (Fl-2)
Jurkat T cells
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Cleaved PARP Expression in Formalin-fixed,
Parafin-embedded Rat Lymph Node
Isotype control (mIgG1)
Cleaved PARP
Cleavage Specific PARP Antibody
Monoclonal (clone F21-852)
Isotype mouse IgG1
Biotin conjugated
Cleaved PARP, high mag
Apoptosis Marker PARP is a Caspase substrate
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PARP Expression in Human, Mouse and Rat Lymph
Node
Isotype Controls
Apoptosis Marker PARP is a Caspase substrate
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BrdU (bromodeoxyuridine) Incorporation Assay
BrdU analog of the DNA precursor, thymidine
Incorporated into newly synthesized DNA DNA
synthesis takes place as cells pass through S
phase Measure levels of cell-associated BrdU by
flow cytometry Detect with BrdU-FITC
mAb -Recognizes BrdU in single stranded
DNA gt Denature DNA with Dnase Concurrently
measure levels of various surface or
intracellular proteins
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Camptothecin-Induced Apoptosis in Jurkat Cells
0 hr
SSC-H
BrdU-FITC
Relative Cell Number
BrdU-FITC
4 hr (4 mM)
FSC-H
Active Caspase-3-PE
Active Caspase-3-PE
7-AAD
Camptothecin prevented cells from synthesizing DNA
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Application of Apoptosis Technology Is
apoptosis associated with Pervanadate-induced
STAT1 activation (20 min) in U937Cells?
Active Caspase-3-PE
Cleaved PARP-PE
Phospho-Stat1-Alexa Fluor 647
Apoptosis Marker PARP is a Caspase substrate
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Apoptotic Sample
Cells
Flow
Fixed
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Apoptosis Signaling
FasL
Receptor
Mitochondrial
Camptothecin
Staurosporine
Fas
FADD
DNA damage
?
Pro-caspase8
ZVAD
Active caspase8
Active Bid
Mitochondria
Pro-caspase9 Apaf-1 Cyt c
ZVAD
Active caspase9
Pro-caspase3 (6, 7)
ZVAD
Active caspase3 (6, 7)
DEATH
Massive substrate cleavage
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Mitoscreen (JC-1) Monitor Mitochondrial Membrane
Potential (Dy)

• Energy is released during oxidation reactions in
  the mitochondrial respiratory chain
• Stored as a negative electrochemical gradient
  Polarized Dy
• Apoptosis is often associated with a decrease in
  Dy Depolarized Dy
• JC-1 1st J-aggregate-forming cationic Dy
  sensitive dye
• J-aggregates form when the Dy is polarized
• JC-1 Monomers exist when the Dy is depolarized
• JC-1 fluorescence on a flow cytometer
• Fl-1 and Fl-2 channels J-aggregates (healthy
  cell)
• Fl-1 channel monomers (apoptosis indicator)

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JC-1 Setup for BD Flow Cytometers FACSCaliburTM,
FACScanTM, FACSortTM

• Instrument Setup with BD CalibriteTM Beads
• Compensation for FL-1/FL-2 with BD CalibriteTM
  Beads
• Start-up the flow cytometer
• Perform standard instrument operation check
• Prepare tubes of BD CaliBRITETM Beads (unlabeled,
  FITC, PE)
• Launch BD FACSCompTM software
• Run BD FACSCompTM using the lyse/no wash
  procedure

Detailed information contained in BD FACSComp
Software Users Guide and BD CaliBRITE Beads
Package
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JC-1 Staining Protocol
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JC-1 Staining in Control and Apoptotic Cells
Jurkat T Cells
Mouse Thymocytes
Control
Control
R1
R1
R1
R2
R2
R2
JC-1 (Fl-2)
Staurosporine
Camptothecin
Fas mAb
R1
R1
R1
R2
R2
R2
JC-1 (Fl-1)
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JC-1 Staining in Mouse Thymocyte Subpopulations
Gated on CD4/CD8
R6
R5
R4
R3
Control
JC-1 (FL-2)
CD8-APC
Fas mAb
CD4-PerCP
JC-1 (FL-1)
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Caspase-Signaling Mitochondrial Depolarization
Active Caspase-3
Cleaved PARP
JC-1
1
95
Control
1
5
JC-1 (Fl-2)
Cell Count
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Camptothecin4 µM, 4 h
40
64
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PE (Fl-2)
JC-1 (Fl-1)
Jurkat T cells
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Comparison of Annexin V, Active Caspase-3,
Cleaved PARP, and JC-1

• Annexin V
• Surface marker established for apoptosis
  validation
• Detect membrane changes associated with apoptosis
  
• Quick assay
• Uses live cells without fixation
• Active Caspase-3 and PARP
• Intracellular markers, established for apoptosis
  validation
• Executioner of apoptosis (Active caspase-3)
• Caspase substrate (Cleaved PARP)
• Uses fixed cells
• JC-1
• Elucidate mitochondrial changes
• Compatible with antibodies to surface markers
• Not compatible with Annexin V or fixation (Active
  caspase-3)

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Selecting and Analyzing Apoptosis Assays

• Measuring apoptosis through caspase activation
  and other mechanisms
• Biology and techniques
• Measuring caspase inhibition
• Inhibitors as tools to protect cells from
  apoptosis/promote cell survival
• Mitochondrial changes during caspase inhibition
• Inhibitors as tools to dissect apoptotic pathways
• Emerging apoptosis technology CBA

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Apoptosis Signaling
FasL
Receptor
Mitochondrial
Camptothecin
Staurosporine
Fas
FADD
DNA damage
?
Pro-caspase8
ZVAD
Active caspase8
Active Bid
Mitochondria
Pro-caspase9 Apaf-1 Cyt c
ZVAD
Active caspase9
Pro-caspase3 (6, 7)
ZVAD
Active caspase3 (6, 7)
DEATH
Massive substrate cleavage
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Cell Permeable Inhibitors as Markers of Caspase
Activation
VAD-FMK Cell permeable peptide inhibitor, binds
with varying affinities to most
caspases FAM-VAD-FMK and SR-VAD-FMK derivatives
FAM-VAD-FMK
FAM-VAD-FMK
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FAM-VAD-FMK Marker for Caspase Activation
Jurkat T cells (unfixed)
Camptothecin
(4µM, 4 h)
Control
SSC-H
FSC-H
Cell Count
FAM-VAD-FMK
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Selecting and Analyzing Apoptosis Assays

• Measuring apoptosis through caspase activation
  and other mechanisms
• Biology and techniques
• Measuring caspase inhibition
• Inhibitors as tools to protect cells from
  apoptosis/promote cell survival
• Mitochondrial changes during caspase inhibition
• Inhibitors as tools to dissect apoptotic pathways
• Emerging apoptosis technology CBA

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BD APO-Block A Cell Culture Grade Caspase
Inhibitor
Broad spectrum caspase inhibitor Propriety
peptidomimetic compound Cell culture
grade Sterile Media supplement Potential
Applications Promote cell survival Inhibit
apoptosis Promote cell preservation recovery
from cryogenic storage Tool for
Dissecting/Defining Signaling Mechanisms Separate
caspase activity from that of other cell death
pathways
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Effect of BD APO-Block on Spontaneous Caspase-3
Activation in Mouse Thymocytes
Control
Control
APO-Block
Control
APO-Block
0 h
24 h
48 h
M1
M1
M1
Cell Number
M2
M1
M1
M2
M2
M2
M2
Active Caspase-3PE
PI
Annexin V-FITC
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Effect of BD APO-Block on Caspase Signaling
Caspase-3
Caspase-7
Cleaved D4-GDI
Caspase-8
Caspase-9
Cleaved PARP
D4-GDI
Control (0 h)
Control (24 h)
BD APO-Block (24 h)
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Viability of Mouse Thymocyte Cultures
100
80
60
Cell Number ( of Control)
40
20
0
24
48
0
Time (h)
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Spontaneous Mouse Apoptosis and Inhibition
Mouse Thymocytes
Caspase Inhibitor (BD APO-Block)
Control
0, 24, 48 hr culture
Cytofix/Cytoperm
Annexin V-FITC
JC-1
Active Caspase-3-PE
Flow Cytometry
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Effect of Caspase Inhibitor on Spontaneous
Apoptosis
24 hr
48 hr
0 hr
Control
Treated
Treated
Control
Control
PI
Annexin V-FITC
RelativeCell Number
Active Caspase-3-PE
JC-1 (FL-2)
JC-1 (FL-1)
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Apoptosis Signaling
FasL
Receptor
Mitochondrial
Camptothecin
Staurosporine
Fas
FADD
DNA damage
?
Pro-caspase8
ZVAD
Active caspase8
Active Bid
Mitochondria
Pro-caspase9 Apaf-1 Cyt c
ZVAD
Active caspase9
Pro-caspase3 (6, 7)
ZVAD
Active caspase3 (6, 7)
DEATH
Massive substrate cleavage
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Selecting and Analyzing Apoptosis Assays

• Measuring apoptosis through caspase activation
  and other mechanisms
• Biology and techniques
• Measuring caspase inhibition
• Inhibitors as tools to protect cells from
  apoptosis/promote cell survival
• Mitochondrial changes during caspase inhibition
• Inhibitors as tools to dissect apoptotic pathways
• Emerging apoptosis technology CBA

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What is a Cytometric Bead Array (CBA)?
ELISA
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Overview of the CBA Assay Configuration
Capture Antibody
Capture Bead



Antigen of Interest
Fluorescent Detector Antibody
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Beads Provide a Flexible Platform
Beads provide an expandable assay platform for
use with a flow cytometer
Multiple sizes
Different intensities
Different colors with different intensities
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3 Bead Apoptosis CBA Assay
Y
Y
Y
Y
Y
Y
Y
Y
Active Caspase-3
Y
Y
Y
Y
Y
DETECTOR ANTIBODY
Wash
Y
Y
Y
Y
Y
Y
Y
Bcl-2
Read on Flow Cytometer
Y
Y
Y
Y
Cleaved PARP
LYSATE OR SUPERNATANT
BEADS
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Cytometric Bead Array Analysis of Cell Lysates
Bcl-2
Cleaved PARP
Active Caspase-3
U937
MFI
MCF-7
Protein (units)
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Apoptosis Signaling
FasL
Receptor
Mitochondrial
Camptothecin
Staurosporine
Fas
FADD
DNA damage
?
Pro-caspase8
ZVAD
Active caspase8
Active Bid
Mitochondria
Pro-caspase9 Apaf-1 Cyt c
ZVAD
Active caspase9
Pro-caspase3 (6, 7)
ZVAD
Active caspase3 (6, 7)
DEATH
Massive substrate cleavage
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CBA
Active Caspase-3
Standard Curves
Data Examples
Control
Control
Jurkat
Jurkat Camptothecin
MCF-7 Camptothecin
Camptothecin
Camptothecin
MFI
FL3-H
10,000 ng
3000 ng
Protein (ng)
FL2-H
Western Blots
MCF-7
Jurkat
Camptothecin
Camptothecin
Control
Control
pAb
mAb
F21-852
C2-10
F21-852
C2-10
116 kDa
32 kDa
85 kDa
17 kDa
C210 Full Length/Cleaved PARP F21-852 Cleaved
PARP
Pro/Active Caspase-3 pAb
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Cytometric Bead Array Analysis of Cell Lysates
Bcl-2
Cleaved PARP
Active Caspase-3
U937
MFI
MCF-7
Protein (units)
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Apoptosis Techniques Summary

• Techniques
• Flow Cytometry
• CBA
• Western Blot
• Immunohistochemistry

• Biology
• Annexin V
• Membrane changes
• APO-BRDU
• DNA fragmentation
• Active Caspase-3
• Apoptosis signaling
• Cleaved PARP
• Caspase substrate
• JC-1
• Mitochondrial changes
• FMKs
• Apoptosis inhibitors

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Contributors 1993-2003
Many Colleagues and Friends who have worked at BD
Biosciences Pharmingen over the last decade
Robert Balderas, VP Research and Development