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Flow Cytometric Analysis Intracellular and Nuclear Antigens T. Vincent Shankey, Ph.D. Systems Research/ Life Sciences Division Beckman Coulter, Inc. Miami,

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Presentation on theme: "Flow Cytometric Analysis Intracellular and Nuclear Antigens T. Vincent Shankey, Ph.D. Systems Research/ Life Sciences Division Beckman Coulter, Inc. Miami,"— Presentation transcript:

1 Flow Cytometric Analysis Intracellular and Nuclear Antigens T. Vincent Shankey, Ph.D. Systems Research/ Life Sciences Division Beckman Coulter, Inc. Miami, FL

2 Intracellular Antigen Analysis Fixation Should Maintain the Cell in its Pre-Fixed State Fixation Should Maintain the Cell in its Pre-Fixed State – Optimal Epitope Expression – Epitope Localization – Stability – Reproducibility – Simplicity

3 Cell Fixation and Permeabilization

4 Intracellular Antigen Analysis Cell Fixation/Permeabilization Whole Cell Techniques Cross-linking fixatives (Formaldehyde) Dehydrating/Denaturing agents (Alcohols) Cross-linking Temp x Time x Concentration Alcohols 4 deg, C) Essentially Instantaneous (“incubate for at least 1 hr…”) Cytoplasmic/Nuclear Membrane Permeabilization Detergents (NP-40, TX-100, Saponin) Alcohols

5 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%)

6 Intracellular Antigen Analysis – Tissue culture cells Cells growing in suspension Cells growing in suspension Attached cells 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) Cell suspensions (blood, bone marrow, FNA, etc) Solid Tissues 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)

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8 Measurement of Cell Signaling

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10 Measurement of Cell Signaling Tissue Culture Cells/Research

11 Measurement of P-STAT5 in Chronic Myeloid Leukemia Cell Line Bcr/Abl Kinase STAT5 STI571 Proliferation (Cyclin D1) Apoptosis (Bcl-X L ) CRKL X X X X ERK X ?

12 Jacobberger, et al. Cytometry 54A;75-88, 2003 Effect of Fixative Concentration on P-STAT5 Expression In K562 Cells

13 Krutzik and Nolan Cytometry 55A;61-70, 2003 Impact of Different Fixation/Permeabilization Techniques On the Expression of Phospho-Specific Intracellular Epitopes

14 Negative Controls 1.No 1 o Antibody (Indirect staining) 2.No Antibody (Direct staining) 3.Negative cell controls a.Cells not expressing Ag b.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

15 Flow Cytometric Assay for STI571 Inhibition of Bcr/Abl using Antibodies to P-STAT5 K562 cell line Jacobberger, et al. Cytometry 54A;75-88, 2003

16 Negative Controls Determination of Negative Staining Using Targeted Inhibitors or Internal Negative Control Cell Populations Jacobberger, et al. Cytometry 54A;75-88, 2003

17 Antibody Validation Western Blot Analysis Flow Cytometry – Use of Appropriate Pos and Neg Controls Fluorescence Microscopy (Ag localization)

18 Validation of STAT5 Phospho-(Tyrosine 694) Antibody STAT5 P-STAT5 Western Blots UntreatedSTI Treated P-STAT5 Flow Cytometry ug P-STAT5/100ul Mean Fluorescence Intentity Untreated + STI

19 Measurement of Cell Cycle Proteins Tissue Culture Cells

20 2% 0.2% 17% S-phase = 51% G 2 /M = 13.5% DNA Content (DAPI) (D) P-H3 Alexa 647 DNA Content (DAPI) (D) Cyclin A2 PE (D) P-H3 Alexa 647 Cyclin A2 PE DNA Content plus Cell Cycle Associated Proteins Cell Cycle Workshop, Bangalore Jan 2011

21 P-H3 Alexa 647 Cyclin A2 PE GatePercent of G 2 /M Events G36% (includes G 2 ) H0.24% I1.40% J0.32% K0.35% L0.35% M27% (includes late S)

22 (D) P-H3 Alexa 647 Cyclin A2 PE Prometaphase Metaphase G2 Anaphase Telophase Cytokenesis G 2 /M defined by Flow Cytometry

23 DNA Content plus Cell Cycle Associated Proteins G 2 D M D G 1 A G 1 D Data acquired on Gallios/3 laser CV D =3.0 Cyclin A2-PE DAPI P-H3 Alexa 647 Cyclin A2-PE P-H3 Alexa 647

24 G 1 D S-G 2 D Early S D Mitotic D Early S An S-G 2 An Mitotic An G 1 A

25 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

26 PMA

27 Control P-Erk-Alexa 488 FA/Triton X uM PMA FA/TX/MeOH Control 40 uM PMA P-Erk-Alexa 488

28 Impact of Fixation/Permeabilization Techniques on Light Scatter Signatures of WBC Method DifferenceSEDFp-value Tukey-Kramer p-value Q-Prep™ vs F/TX < Q-Prep™ vs F/TX/MeOH < F/TX vs F/TX/MeOH X2X2 Y2Y2 FS SS Distance (r) = Fisher Distance =

29 Estimates of Total Bias for WBC Comparing Results of Flow Cytometric Analysis with CBC CBC values determined by LH750 TM. 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 Granulocytes Monocytes FS CD45 Side Scatter

30 Intensity of CD Marker Expression on Different WBC Populations using Different Whole Blood Preparation Techniques

31 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

32 Collaborators ACCG/Cytometry Consortium David Hedley/Sue Chow /Qing Chang– Ontario Cancer Institute, UHN, Toronto, Ont. Chuck Goolsby/James Marvin – Northwestern University, Chicago, IL Jim Jacobberger/Phil Woost - Case Western Reserve Univ, Cleveland, OH Beckman Coulter Patty Grom, Lilly Lopez – Advanced Technology/Systems Research Meryl Forman & Co (Ltd) – Advanced Technology Bob Zigon/Ernie Anderson – Kaluza Software Development


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