Post on 11-Jan-2016
description
Flow Cytometric Analysis Intracellular and Nuclear Antigens
T. Vincent Shankey, Ph.D.Systems Research/ Life Sciences Division
Beckman Coulter, Inc.Miami, FL
Vincent.Shankey@Coulter.com
Intracellular Antigen Analysis
• Fixation Should Maintain the Cell in its Pre-Fixed State– Optimal Epitope Expression– Epitope Localization– Stability– Reproducibility– Simplicity
Cell Fixation and Permeabilization
Intracellular Antigen Analysis
Cell Fixation/PermeabilizationWhole Cell Techniques
Cross-linking fixatives (Formaldehyde)Dehydrating/Denaturing agents (Alcohols)
Cross-linkingTemp x Time x Concentration
Alcohols (generally @ 4 deg, C)Essentially Instantaneous (“incubate for at least 1 hr…”)
Cytoplasmic/Nuclear Membrane PermeabilizationDetergents (NP-40, TX-100, Saponin)Alcohols
Permeabilization using Saponin • Earliest reports (?) using Saponin for intracellular staining and flow
cytometry:– Andersson U. and B. Sander, Immunol Lett 20; 115, 1989
(Intracellular IL-2 plus surface CD4/CD8 staining)– Bardales R.H., et al, J Histochem Cytochem 37: 83, 1989
• Many commercial “fix and perm” kits use Saponin (IntraPrep™, Fix&Perm™)
• Saponin is a detergent extracted from the bark of Quillaja trees (biologic activity varies from lot to lot)– Interacts with membrane cholesterol to form pores– Pores are reversible (added cholesterol, temperature)
• Cytoplasmic/nuclear antibody staining should be performed at room temperature, with cholesterol-free buffer (no NCS), and buffer should contain low concentration of saponin (0.01 to 0.05%)
Intracellular Antigen Analysis
– Tissue culture cells• Cells growing in suspension• Attached cells
– Potential problem of alteration of cell surface and/or intracellular epitopes (e.g. FAK-linked pathways)
– Clinical Samples• Cell suspensions (blood, bone marrow, FNA, etc)• Solid Tissues
– Isolation of single cell suspension (yield, recovery, alteration of cell surface and/or intracellular epitopes
(see Hitchcock, CL and Ensley, JF, pp93-110, in Clinical Flow Cytometry; Principles and Application. Williams&Wilkins, Baltimore, 1993)
Measurement of Cell Signaling
Measurement of Cell Signaling
Tissue Culture Cells/Research
Measurement of P-STAT5 in Chronic Myeloid Leukemia Cell Line
Bcr/Abl Kinase
STAT5
STI571
Proliferation(Cyclin D1)
Apoptosis (Bcl-XL)
CRKLX
X
X
XERK
X
?
Jacobberger, et al. Cytometry 54A;75-88, 2003
Effect of Fixative Concentration on P-STAT5 Expression In K562 Cells
Krutzik and Nolan Cytometry 55A;61-70, 2003
Impact of Different Fixation/Permeabilization TechniquesOn the Expression of Phospho-Specific Intracellular Epitopes
Negative Controls
1. No 1o Antibody (Indirect staining)
2. No Antibody (Direct staining)
3. Negative cell controls a. Cells not expressing Agb. Ag neg cell subpopulation (e.g. M cells and P-STAT5)c. Gene knockout or siRNA
4. Inhibition of Ag Expression (e.g. Gleevec)
5. Isoclonic Controls
6. Isotype Controls
1 10 1000
250
500
750
1000 PositiveSecondary OnlyImatinibUnstained
Fluorescence
Cel
l Nu
mb
er
Flow Cytometric Assay for STI571 Inhibition ofBcr/Abl using Antibodies to P-STAT5
K562 cell line
Jacobberger, et al. Cytometry 54A;75-88, 2003
Negative Controls
Determination of Negative Staining Using Targeted Inhibitors or Internal Negative Control Cell Populations
Jacobberger, et al. Cytometry 54A;75-88, 2003
Antibody Validation
Western Blot Analysis
Flow Cytometry – Use of Appropriate Pos and Neg Controls
Fluorescence Microscopy (Ag localization)
Validation of STAT5 Phospho-(Tyrosine 694)Antibody
STAT5P-STAT5
Western Blots
250
150
100
75
50
Untreated STI Treated
P-STAT5
Titration of Rab P-STAT5-Alexa 488
0
5
10
15
20
25
0 0.1 0.2 0.5 1 2
ug P-STAT5/100 ul
Fluore
scen
ce
UnTx
STI571 2uM 22hr
Flow Cytometry
ug P-STAT5/100ul
Mea
n F
luo
resc
ence
Inte
nti
ty
Untreated
+ STI
Measurement of Cell Cycle Proteins
Tissue Culture Cells
2%
0.2%
17%
S-phase = 51%
G2/M = 13.5%
DNA Content (DAPI) DNA Content (DAPI)
(D)
P-H
3 A
lexa
647
DNA Content (DAPI)
(D)
Cyc
lin A
2 PE
(D)
P-H
3 A
lexa
647
Cyclin A2 PE
DNA Content plus Cell Cycle Associated Proteins
Cell Cycle Workshop, Bangalore Jan 2011
P-H
3 A
lexa
647
Cyclin A2 PE
Gate Percent of G2/M Events
G 36% (includes G2)H 0.24%I 1.40%J 0.32%K 0.35%L 0.35%M 27% (includes late S)
(D)
P-H
3 A
lexa
647
Cyclin A2 PE
Prometaphase
Metaphase
G2
Anaphase
Telophase
Cytokenesis
G2/M defined by Flow Cytometry
DNA Content plus Cell Cycle Associated Proteins
0 200 400 600 800 1000FL9-A (x 1000)
01
02
03
04
05
06
07
0N
um
be
r
100 101 102 103 104 105 106
DAPI
10
01
01
10
21
03
10
41
05
10
6C
yclin
A2
PE
100 101 102 103 104 105 106
DAPI
10
01
01
10
21
03
10
41
05
10
6P
-H3
Ale
xa 6
47
R1
R2
100 101 102 103 104 105 106
Cyclin A2 PE
10
01
01
10
21
03
10
41
05
10
6P
-H3
Ale
xa 6
47
G2 D
M D
G1 A
G1 D
Data acquired on Gallios/3 laser
CVD=3.0
Cyclin A2-PEDAPI
P-H
3 A
lexa
647
Cyc
lin
A2-
PE
P-H
3 A
lexa
647
G1 D
S-G2 D
Early S D
Mitotic D
Early S An
S-G2 An
Mitotic An
G1 A
Measurement of Cell Signaling
Whole Blood/Bone Marrow
• Whole Blood Fixation and Permeabilization Method
• Preservation of Light Scatter and CD Epitopes
• Internal Positive and Negative Control Populations
PMA
Control
P-Erk-Alexa 488
FA/Triton X-100
40 uM PMA
FA/TX/MeOH
Control
40 uM PMA
P-Erk-Alexa 488
Impact of Fixation/PermeabilizationTechniques on Light Scatter Signatures of WBC
Method Difference SE DF p-valueTukey-Kramer p-value
Q-Prep™ vs F/TX -0.435 0.01612 408 <0.0001 <0.0001
Q-Prep™ vs F/TX/MeOH -0.4553 0.01612 408 <0.0001 <0.0001
F/TX vsF/TX/MeOH
0.0203 0.01612 408 0.2092 0.42
X2
Y2
FS
SS
Distance (r) = Fisher Distance =
22 YX
BA
Distance
SDSD
Estimates of Total Bias for WBCComparing Results of Flow Cytometric Analysis with CBC
CBC values determined by LH750TM. Approximate tolerance limits (-----) determined by the CBC are plotted against the determinations for lymphocytes using determinations of WBC populations from individual samples prepared using Q-Prep™ ( ), or F/TX ( ), or F/TX/MeOH ( ).
Lymphocytes
-200
0
200
400
600
800
1000
1200
1400
0 500 1000 1500 2000 2500 3000
Average
Monocytes
-400
-300
-200
-100
0
100
200
300
400
0 200 400 600 800 1000
Average
Granulocytes
-1500
-1000
-500
0
500
1000
1500
1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000
Average
Lymphocytes
Granulocytes
Monocytes
FS
CD45
Side
Sca
tter
Side
Sca
tter
Marker Q-PrepTM F/TX F/TX/MeOH
MFIB SD MFIB SD MFIB SD
LymphocytesA
CD45 353.6 110. 201.7 76.0 275.6 56.9
CD3 123.1 29.7 126.6 20.5 124.9 18.7
CD19 48.3 12.0 16.6 84.2 16.3 1.9
MonocytesA
CD45 224.9 66.8 260 108.8 351.3 62.7
CD13 84.1 45.6 41.5 19.2 41.9 18.3
CD14 81.4 28.9 103.7 21.6 21.4 15.7
CD33 44.0 20.9 38.9 10.7 20.1 9.4
GranulocytesA
CD45 69.9 20.9 127.0 56.9 185.7 37.8
CD13 53.3 20.5 51.4 12.0 53.0 6.0
CD33 12.1 2.7 12.2 7.6 12.0 7.9
Intensity of CD Marker Expression on Different WBC Populations using Different Whole Blood Preparation Techniques
Advantages of Whole Blood Sampling for Signal Transduction Pathway Analysis
• Sample Processing Speed– No cell separation step(s)– Rapid fixation minimizes potential for spontaneous de-
phosphorylation of target epitopes (cytoplasmic phosphatases)
– Ideal for use in clinical setting• Minimal Cell Loss
– Cell separation techniques can deplete specific cell types• Keeps Target Cell Populations in Contact with Pathway
Inhibitors (Targeted Therapeutics)– Rapid loss/reversal of in vivo pathway inhibition after
removal of cells from serum
CollaboratorsACCG/Cytometry Consortium
David Hedley/Sue Chow /Qing Chang– Ontario Cancer Institute, UHN, Toronto, Ont.
Chuck Goolsby/James Marvin – Northwestern University, Chicago, ILJim Jacobberger/Phil Woost - Case Western Reserve Univ, Cleveland, OH
Beckman Coulter
Patty Grom, Lilly Lopez – Advanced Technology/Systems ResearchMeryl Forman & Co (Ltd) – Advanced TechnologyBob Zigon/Ernie Anderson – Kaluza Software Development