Single‐Cell Phospho‐Protein Analysis by Flow Cytometry

Kenneth R. Schulz1, Erika A. Danna1, Peter O. Krutzik1, Garry P. Nolan1

1 Stanford University, Stanford, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.17
DOI:  10.1002/0471142735.im0817s96
Online Posting Date:  February, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This protocol describes methods for monitoring intracellular phosphorylation‐dependent signaling events on a single‐cell basis. This approach measures cell signaling by treating cells with exogenous stimuli, fixing cells with formaldehyde, permeabilizing with methanol, and then staining with phospho‐specific antibodies. Thus, cell signaling states can be determined as a measure of how cells interact with their environment. This method has applications in clinical research as well as mechanistic studies of basic biology. In clinical research, diagnostic or drug efficacy information can be retrieved by discovering how a disease affects the ability of cells to respond to growth factors. Basic scientists can use this technique to analyze signaling events in cell lines and human or murine primary cells, including rare populations, like B1 cells or stem cells. This technique has broad applications bringing standard biochemical analysis into primary cells in order to garner valuable information about signaling events in physiologic settings. Curr. Protoc. Immunol. 96:8.17.1‐8.17.20. © 2012 by John Wiley & Sons, Inc.

Keywords: phosphorylation; signaling; flow cytometry; FACS; intracellular staining

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Measuring Phosphorylation Events at the Single‐Cell Level by Flow Cytometry
  • Alternate Protocol 1: Phospho‐Protein Analysis in Murine Primary Lymphocytes Stimulated with Cytokines or Growth Factors
  • Alternate Protocol 2: Phospho‐Protein Analysis in Human Primary Lymphocytes Stimulated with Cytokines or Growth Factors
  • Support Protocol 1: Optimization of Surface Marker Staining for Phospho‐Specific Flow Cytometry
  • Reagents And Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Measuring Phosphorylation Events at the Single‐Cell Level by Flow Cytometry

  Materials
  • Cell line of interest (e.g., U937 cells)
  • Tissue culture medium (see recipe)
  • Stimuli of interest (e.g., human IL‐4 and human IFN‐γ)
  • 16% paraformaldehyde in water (PFA, EM‐grade; Electron Microscopy Sciences)
  • 100% methanol, 4°C
  • FACS buffer (see recipe)
  • Directly labeled phospho‐specific antibodies (e.g., Ax488‐conjugated anti‐Stat1(pY701) and Ax647‐conjugated anti‐Stat6(pY641))
  • 5‐ml polystyrene FACS tubes (BD Falcon)
  • 37°C, 5% CO 2 incubator
  • Beckman Coulter Allegra 64R centrifuge with GH 3.8A rotor (or equivalent)
  • Flow cytometer with 488‐ and 633‐nm laser lines (e.g., Becton Dickinson FACSCalibur)

Alternate Protocol 1: Phospho‐Protein Analysis in Murine Primary Lymphocytes Stimulated with Cytokines or Growth Factors

  Materials
  • Murine tissue of interest (e.g., spleen)
  • MEM/FBS (see recipe)
  • Tissue culture medium (see recipe)
  • Stimuli of interest (e.g., murine IL‐10)
  • 16% paraformaldehyde in water (PFA, EM‐grade; Electron Microscopy Sciences)
  • 100% methanol, 4°C
  • FACS buffer (see recipe)
  • Directly labeled phospho‐specific antibodies (Ax647‐conjugated anti‐Stat3(pY705))
  • Directly labeled surface marker antibodies (e.g., PE‐conjugated anti‐mouse TCRβ and PerCP‐Cy5.5‐conjugated anti‐mouse B220 at 0.2 mg/ml; BD Pharmingen)
  • Frosted microscope slides or syringe plungers from 1‐ or 3‐ml syringes
  • 70‐µm nylon cell strainers
  • 50‐ml polypropylene tubes
  • 37°C, 5% CO 2 incubator
  • 5‐ml FACS tubes
  • Flow cytometer with 488‐ and 633‐nm laser lines (e.g., Becton Dickinson FACSCalibur)

Alternate Protocol 2: Phospho‐Protein Analysis in Human Primary Lymphocytes Stimulated with Cytokines or Growth Factors

  • Primary human cells of interest (e.g., human peripheral blood mononuclear cells, PBMC), frozen or fresh
  • Human tissue culture medium (see recipe), 37°C
  • Stimuli of interest (e.g., human IL‐10 and human IFN‐γ)
  • Directly labeled phospho‐specific antibodies (e.g., Ax488‐conjugated anti‐Stat3(pY705) and Ax647‐conjugated anti‐Stat1(pY701), BD Biosciences)
  • Directly labeled surface marker antibodies (e.g., PE‐conjugated anti‐human CD33 clone P67.6, PE‐Cy7‐conjugated anti‐human CD4 clone RPA‐T4, PerCP‐Cy5.5‐labeled anti‐human CD20 clone H1, and Ax700‐conjugated anti‐human CD3 clone UCHT‐1; BD Biosciences)
  • 37°C water bath
  • 15‐ml polypropylene tubes
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Chow, S., Patel, H., and Hedley, D.W. 2001. Measurement of MAP kinase activation by flow cytometry using phospho‐specific antibodies to MEK and ERK: Potential for pharmacodynamic monitoring of signal transduction inhibitors. Cytometry 46:72‐78.
   Chow, S., Hedley, D., Grom, P., Magari, R., Jacobberger, J.W., and Shankey, T.V. 2005. Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67:4‐17.
   Danna, E.A. and Nolan, G.P. 2006. Transcending the biomarker mindset: Deciphering disease mechanisms at the single‐cell level. Curr. Opin. Chem. Biol. 10:20‐27.
   Fleisher, T.A., Dorman, S.E., Anderson, J.A., Vail, M., Brown, M.R., and Holland, S.M. 1999. Detection of intracellular phosphorylated STAT‐1 by flow cytometry. Clin. Immunol. 90:425‐430.
   Grammer, A.C., Fischer, R., Lee, O., Zhang, X., and Lipsky, P.E. 2004. Flow cytometric assessment of the signaling status of human B lymphocytes from normal and autoimmune individuals. Arthritis Res. Ther. 6:28‐38.
   Hale, M.B. and Nolan, G.P. 2006. Phospho‐specific flow cytometry: Intersection of immunology and biochemistry at the single‐cell level. Curr. Opin. Mol. Ther. 8:215‐224.
   Irish, J.M., Hovland, R., Krutzik, P.O., Perez, O.D., Bruserud, O., Gjertsen, B.T., and Nolan, G.P. 2004. Single cell profiling of potentiated phospho‐protein networks in cancer cells. Cell 118:217‐228.
   Irish, J.M., Anensen, N., Hovland, R., Skavland, J., Borresen‐Dale, A.L., Bruserud, O., Nolan, G.P., and Gjertsen, B.T. 2006a. Flt3 Y591 duplication and Bcl‐2 overexpression are detected in acute myeloid leukemia cells with high levels of phosphorylated wild‐type p53. Blood 109:2589‐2596.
   Irish, J.M., Czerwinski, D.K., Nolan, G.P., and Levy, R. 2006b. Altered B‐cell receptor signaling kinetics distinguish human follicular lymphoma B cells from tumor‐infiltrating nonmalignant B cells. Blood 108:3135‐3142.
   Irish, J.M., Czerwinski, D.K., Nolan, G.P., and Levy, R. 2006c. Kinetics of B cell receptor signaling in human B cell subsets mapped by phosphospecific flow cytometry. J. Immunol. 177:1581‐1589.
   Juan, G., Traganos, F., James, W.M., Ray, J.M., Roberge, M., Sauve, D.M., Anderson, H., and Darzynkiewicz, Z. 1998. Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis. Cytometry 32:71‐77.
   Krutzik, P.O. and Nolan, G.P. 2003. Intracellular phospho‐protein staining techniques for flow cytometry: Monitoring single cell signaling events. Cytometry A 55:61‐70.
   Krutzik, P.O. and Nolan, G.P. 2006. Fluorescent cell barcoding in flow cytometry allows high‐throughput drug screening and signaling profiling. Nat. Methods 3:361‐368.
   Krutzik, P.O., Clutter, M.R., and Nolan, G.P. 2005a. Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry. J. Immunol. 175:2357‐2365.
   Krutzik, P.O., Hale, M.B., and Nolan, G.P. 2005b. Characterization of the murine immunological signaling network with phosphospecific flow cytometry. J. Immunol. 175:2366‐2373.
   Mackeigan, J.P., Murphy, L.O., Dimitri, C.A., and Blenis, J. 2005. Graded mitogen‐activated protein kinase activity precedes switch‐like c‐Fos induction in mammalian cells. Mol. Cell. Biol. 25:4676‐4682.
   Perez, O.D. and Nolan, G.P. 2002. Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat. Biotechnol. 20:155‐162.
   Perez, O.D., Krutzik, P.O., and Nolan, G.P. 2004. Flow cytometric analysis of kinase signaling cascades. Methods Mol. Biol. 263:67‐94.
   Pollice, A.A., McCoy, J.P. Jr., Shackney, S.E., Smith, C.A., Agarwal, J., Burholt, D.R., Janocko, L.E., Hornicek, F.J., Singh, S.G., and Hartsock, R.J. 1992. Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins. Cytometry 13:432‐444.
   Ricciardi, M.R., McQueen, T., Chism, D., Milella, M., Estey, E., Kaldjian, E., Sebolt‐Leopold, J., Konopleva, M., and Andreeff, M. 2005. Quantitative single cell determination of ERK phosphorylation and regulation in relapsed and refractory primary acute myeloid leukemia. Leukemia 19:1543‐1549.
   Roederer, M. 2001. Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats. Cytometry 45:194‐205.
   Sachs, K., Perez, O., Pe'er, D., Lauffenburger, D.A., and Nolan, G.P. 2005. Causal protein‐signaling networks derived from multiparameter single‐cell data. Science 308:523‐529.
   Schaap, A., Fortin, J.F., Sommer, M., Zerboni, L., Stamatis, S., Ku, C.C., Nolan, G.P., and Arvin, A.M. 2005. T‐cell tropism and the role of ORF66 protein in pathogenesis of varicella‐zoster virus infection. J. Virol. 79:12921‐12933.
   Tazzari, P.L., Cappellini, A., Bortul, R., Ricci, F., Billi, A.M., Tabellini, G., Conte, R., and Martelli, A.M. 2002. Flow cytometric detection of total and serine 473 phosphorylated Akt. J. Cell. Biochem. 86:704‐715.
   Tong, F.K., Chow, S., and Hedley, D. 2006. Pharmacodynamic monitoring of BAY 43‐9006 (Sorafenib) in phase I clinical trials involving solid tumor and AML/MDS patients, using flow cytometry to monitor activation of the ERK pathway in peripheral blood cells. Cytometry B Clin. Cytom. 70:107‐114.
   Uzel, G., Frucht, D.M., Fleisher, T.A., and Holland, S.M. 2001. Detection of intracellular phosphorylated STAT‐4 by flow cytometry. Clin. Immunol. 100:270‐276.
   Van Meter, M.E., Diaz‐Flores, E., Archard, J.A., Passegue, E., Irish, J.M., Kotecha, N., Nolan, G.P., Shannon, K., and Braun, B.S. 2006. K‐RasG12D expression induces hyperproliferation and aberrant signaling in primary hematopoietic stem/progenitor cells. Blood 109:3945‐3952.
   Zampieri, C.A., Fortin, J.F., Nolan, G.P., and Nabel, G.J. 2006. The ERK mitogen‐activated protein kinase pathway contributes to ebola glycoprotein‐induced cytotoxicity. J. Virol. 81:1230‐1240.
   Zell, T. and Jenkins, M.K. 2002. Flow cytometric analysis of T cell receptor signal transduction. Sci. STKE. 2002:PL5.
   Zell, T., Khoruts, A., Ingulli, E., Bonnevier, J.L., Mueller, D.L., and Jenkins, M.K. 2001. Single‐cell analysis of signal transduction in CD4 T cells stimulated by antigen in vivo. Proc. Natl. Acad. Sci. U.S.A. 98:10805‐10810.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library