This document discusses the use of activation-state specific antibodies for analyzing cellular signaling using imaging cytometry techniques like immunofluorescence and high content analysis. It provides examples of how these antibodies can be used to study phosphorylation events, apoptosis, and validate optimal antibody concentrations. The document also discusses the potential for using these antibodies in flow cytometry applications like clinical assays to study disease signaling pathways.

1. Analysis of Cellular Signaling using
Activation-State Specific Antibodies
Greg Innocenti
Cell Signaling Technology
December 19, 2006

2. Total Antibodies
Untreated Wnt 5’ Whole Blood
HeLa Cells
CD5
β-Catenin (C-term)
CD13
LY294002 Insulin
Side Scatter
C2C12 Cells
Akt (pan) (11E7)
CD4

3. Phospho-specific Antibodies
Untreated EGF-Treated
Phospho-EGFR (PE)
EGF Receptor (FITC)
Green = Phospho-EGF Receptor (Tyr1068)
Blue = DRAQ5

4. Caspase-3 Cleavage in Apoptosis
Untreated Staurosporine
TUNEL
Cleaved-Caspase 3
Green = Cleaved Caspase-3 (Asp175) (5A1) RmAb
Red = F-Actin (Phalloidin)
Blue = DRAQ5

5. Antibody Validation for Immunofluorescence/HCA
Part I - Titration to determine Part II - Specificity Testing
optimal dilution/concentration • treat cells with specific
ligands, drugs, inhibitors, etc.
• test antibody on cells that do
and do not express target
• verify expression or treatment
efficacy with another antibody
(same target, or different
target in same pathway)
Confocal Imaging on Chamber Slides
Titration Curve
45000.00 6.00
40000.00
5.00
35000.00
30000.00 4.00
2966
Mean Channel Fluorescence
Signal to Noise Ratio
25000.00
3.00 Control
20000.00
S/N Ratio
15000.00 2.00
10000.00
1.00
5000.00
0.00 0.00
0 5 10 15 20 25 30 35 40 45
Antibody Dilution (ug/ml)

6. Antibody Validation for Immunofluorescence/HCA
[Ab]
Antibody
Isotype
Antibody
1:25
1:50
1:100
1:200
1:400
1:800
16000.00 4.50
14000.00 4.00
12000.00 3.50
3.00
10000.00
Isotype
4694
Mean Channel Fluorescence
2.50
Signal to Noise Ratio
8000.00 Control
2.00
6000.00 S/N Ratio
1.50
4000.00
1.00
2000.00 0.50
0.00 0.00
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Antibody Dilution (ug/ml)
MEK1/2 (red)
actin (green)
DNA (blue)
HeLa cells

7. CST Conjugated Antibodies
Conjugated antibodies helpful for multiplex analyses
CST conjugates are optimized for cytometric applications
• high-quality pre-validated antibodies
• bright photostable Alexa dyes
• F/P trials to ensure bright signal
• antibody titration
• stability tested (accelerated and real-time)
• screened with flow cytometry, HCA, and IF
• lot-to-lot stability

8. Survivin (Alexa488-conjugate)
FAS, TNFa
• inhibits apoptosis and regulates mitosis
• over-expressed in most human cancers
FADD
• expression correlates with both accelerated Caspase-8/10
ER Stress Mitochondria
relapse and chemotherapy resistance
[Ca++] Smac/
P1 Rat Brain Diablo Cyto C
Caspase-12 Survivin
Caspase-9
Caspase-3
Caspase-6 Caspase-7
Lamin A a-Fodrin DFF PARP
CST’s conjugated Survivin antibody is currently being used to screen patient samples

9. The Bigger Picture
Single well immunofluorescence
+ ability to examine subcellular (co)localization in 4-dimension
(XYZt)
- difficult to quantify without specialized software
- imaging is time consuming and data files become massive
High Content Analysis
+ some systems able to analyze localization
+ rapid scanning (comparatively), sensitive, and quantifiable
+ ability to multiplex and dissect various pathways in tandem

10. High Content Analysis
• automated plate-based image analysis
• quantifiable signal intensity and subcellular
1:800
1:100
1:400
1:200
1:50
1:25
localization Untreated
• more predictive of drug activity in a cellular PDGF
p-Erk
environment compared to ELISAs U0126+PDGF
LY/Wort+PDGF
• can be used to determine: Untreated
efficacy and therapeutic dose
PDGF
p-Akt
U0126+PDGF
cell-permeability of drugs LY/Wort+PDGF
potential toxicity (DNA damage, apoptosis,
micronuclei)
downstream effects of drug/target interaction
off-target effects

11. Value of CST Antibodies in HCS
Current platforms have increased colors and resolution in an attempt to
quantify complex events
Example: nuclear translocation of Erk or NFkB
Requirements
• nuclear marker
• total antibody
• high resolution optics
• complex software
• lots of data storage
Using a phospho antibody will eliminate these costly requirements and
speed up the screen (on/off as opposed to determination of localization)
KEY: reliable simple affordable assay with clear robust results

12. Multiple, Parallel Analyses by Cellular Imaging (ICW)
Gleevec/PDGF
U0126/PDGF
LY294002
Untreated
Gleevec
PDGF
PDGF
PMA
blank
p-PDGFR
p-Erk
p-Akt
RTK signaling analysis using LI-COR Odyssey
Untreated Anisomycin UV
p-p38
p-Jnk
p-ATF2
p-H2A.X
DNA damage profiling analysis using LI-COR Odyssey

13. Future Plans: Broad Signal Profiling by HCA
• large antibody panel for profiling screens
• can be used on any cytometric platform
• detection = fluorescent, chromogenic, chemiluminescence
Starved
• up to 96 antibodies per plate
total and phosphorylation-specific antibodies
MAPK, Akt, NFkB, Jak/Stat, etc.
receptor tyrosine kinases (EGFR, VEGFR, FGFR, IGF-IR, cKit)
adaptor proteins and downstream targets EGF
transcription factors
motif antibodies (general serine or tyrosine phosphorylation)
• can be customized for analysis of different biological processes
toxicity
cell cycle/arrest Anisomycin
apoptosis
cell adhesion
96 prediluted cells treated with
CST antibodies compound X
UV

14. Antibody Development: FoxO1 Screening
PI3K inhibitor IGF-1 + serum
(Akt off) (AKT on)
How can we generate antibodies that
work well for cytometric applications?
Screen using cytometric applications
HTS & HCS Platforms
Nuclear Cytoplasmic
nuclear trigger
(half-width intensity)

15. Outline
What are activation state-specific antibodies?
Imaging Cytometry
• immunofluorescence & antibody validation
• antibody conjugation
• high content analysis (HCA)
Flow Cytometry
• protocols
• clinical assays

16. Optimization: Fixation/Permeabilization
CST 2-4% formaldehyde/90% methanol
Fix&Perm Kits 0.25-4% formaldehyde/detergent (triton or saponin)
Krutzik and Nolan (2004) Cytometry 55A:61-70.

17. Fixation/Permeabilization
p-p38
p-Erk
2% Formaldehyde 4% Formaldehyde Commercial Fix&Perm Kit
90% Methanol 0.3% Triton X-100 (aldehyde/saponin)

18. Surface and Signaling Markers
• Methanol diminishes or abolishes signal from some key surface markers
• Many signaling event are very transient so immediate fixation is critical, no
time to prelabel with surface markers, lyse RBCs, or perform FiColl
separations
• Staggered protocol: fix cells with aldehyde to stop all enzymes and preserve
phosphoepitopes, label with surface markers, permeabilize with methanol,
and then label with intracellular signaling antibodies (destroys some
fluorochromes)
• Need a better solution…

19. New Fix & Perm Protocol
Untreated
PMA
4% Formaldehyde + 0.1% Triton X-100 + 50% Methanol

20. Protocol Comparison
4%PFA at RT for 10m
3% PFA at 37ºC for 10m 0.1% Triton X-100 at RT for 30m
90% ice cold MeOH at -20ºC for 10m 50% ice cold MeOH at 4ºC for 10m
CD19 (FITC) CD19 (FITC)

21. Flow Cytometry: Clinical Applications
• Staining of intracellular signaling molecules can be easily
incorporated with traditional cell surface marker labeling
4% Formaldehyde for 10m
0.1 Trinton X-100 ft RT for 30m
50% ice cold MeOH at 4oC for 10m
• Important implications in the Clinic
• Flow Cytometry and Disease-specific Signaling
• Chronic myelogenous leukemia (CML)
• Chronic lymphocytic leukemia (CLL)
• Acute myelogenous leukemia (AML)

22. Bcr/Abl Pathway Profiling by Flow Cytometry
CML cells (K562) + Gleevec
phospho-
Bcr
phospho-
Stat5
cleaved-
Casp3
0 hr 1 hr 24 hr 48 hr