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Flow Cytometric Analysis Intracellular and Nuclear Antigens

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

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Flow Cytometric Analysis Intracellular and Nuclear Antigens

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  1. 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

  2. Intracellular Antigen Analysis • 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 (generally @ 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 • 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)

  7. Measurement of Cell Signaling

  8. Measurement of Cell SignalingTissue Culture Cells/Research

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

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

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

  12. Negative Controls • No 1o Antibody (Indirect staining) • No Antibody (Direct staining) • Negative cell controls • Cells not expressing Ag • Ag neg cell subpopulation (e.g. M cells and P-STAT5) • Gene knockout or siRNA • Inhibition of Ag Expression (e.g. Gleevec) • Isoclonic Controls • Isotype Controls

  13. 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

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

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

  16. STAT5 Flow Cytometry P-STAT5 Western Blots Untreated STI Treated Untreated 250 Mean Fluorescence Intentity 150 + STI 100 75 50 ug P-STAT5/100ul P-STAT5 Validation of STAT5 Phospho-(Tyrosine 694) Antibody

  17. Measurement of Cell Cycle ProteinsTissue Culture Cells

  18. S-phase = 51% G2/M = 13.5% (D) (D) P-H3 Alexa 647 Cyclin A2 PE DNA Content (DAPI) Cyclin A2 PE DNA Content plus Cell Cycle Associated Proteins (D) 2% P-H3 Alexa 647 0.2% 17% DNA Content (DAPI) DNA Content (DAPI) Cell Cycle Workshop, Bangalore Jan 2011

  19. P-H3 Alexa 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)

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

  21. M D G1 D P-H3 Alexa 647 P-H3 Alexa 647 Cyclin A2-PE G2 D G1 A DAPI Cyclin A2-PE CVD=3.0 Data acquired on Gallios/3 laser DNA Content plus Cell Cycle Associated Proteins

  22. Mitotic D Mitotic An S-G2 An S-G2 D Early S D Early S An G1 D G1 A

  23. Measurement of Cell SignalingWhole Blood/Bone Marrow • Whole Blood Fixation and Permeabilization Method • Preservation of Light Scatter and CD Epitopes • Internal Positive and Negative Control Populations

  24. PMA

  25. Control Control FA/Triton X-100 FA/TX/MeOH 40 uM PMA 40 uM PMA P-Erk-Alexa 488 P-Erk-Alexa 488

  26. Impact of Fixation/PermeabilizationTechniques on Light Scatter Signatures of WBC FS Y2 X2 Distance (r) = Fisher Distance = SS

  27. Lymphocytes Side Scatter Monocytes CD45 Side Scatter Granulocytes 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 ( ). FS Estimates of Total Bias for WBCComparing Results of Flow Cytometric Analysis with CBC

  28. 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

  29. 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

  30. 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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