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