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

  •   FigureFigure 8.17.1 Analysis of U937 sample experiment. U937 cells were left unstimulated or stimulated with IFN‐γ, IL‐4, or the combination of IFN‐γ and IL‐4 for 15 min. The cells were then fixed with formaldehyde, permeabilized with methanol, and stained with pStat1 Ax488 and pStat6 Ax647 phospho‐specific antibodies. After staining, cells were run on a FACSCalibur flow cytometer. Shown are one‐dimensional histogram (A) and two‐dimensional dot‐plot (B) analyses of the phospho‐proteins. (A) Each phospho‐protein is analyzed individually with overlaid histograms to reveal the induction of pStat1 by IFN‐γ and pStat6 by IL‐4. Note how the histograms of the two samples stimulated with IFN‐γ or IL‐4 line up (indicated with arrows). Also note that the unstained sample, which represents cellular autofluorescence alone, is slightly lower than unstimulated samples in fluorescence intensity. (B) The two phospho‐proteins are analyzed simultaneously in two‐dimensional plots. Here, note how induction with IFN‐γ or IL‐4 moves the population in the vertical or horizontal direction, respectively, indicating induction of only one phospho‐protein at a time. However, the combination of IFN‐γ and IL‐4 induces both phospho‐proteins, resulting in a shift to the upper right quadrant on the two‐dimensional dot plot.
  •   FigureFigure 8.17.2 Analysis of IL‐10 time course experiment in murine splenocytes. BALB/c splenocytes were stimulated with 50 ng/ml of IL‐10 for various times before fixation in formaldehyde and permeabilization in methanol. The cells were then stained with TCRβ PE, B220 PerCP‐Cy5.5, and pStat3 Ax647 and analyzed on a FACSCalibur flow cytometer. In step 1 (A), lymphocytes are gated by forward and side scatter characteristics to eliminate debris and red blood cells. The resulting lymphocytes are then separated into B and T cell populations based on their surface expression of the B cell marker B220 and the T cell marker TCRβ. In step 2 (B), the phosphorylation of Stat3 at various time points is analyzed in B and T cells in response to IL‐10 stimulation. Note how both cell types achieve maximal phosphorylation at 15 min, with phosphorylation fading slowly over 2 hr to near‐basal states. Also note the stronger induction of Stat3 in B cells relative to T cells in response to IL‐10.
  •   FigureFigure 8.17.3 Analysis of human PBMC experiment. Human PBMC were prepared by Ficoll gradient purification, frozen, and thawed prior to stimulation with 50 ng/ml IFN‐γ or IL‐10. The cells were fixed with formaldehyde prior to permeabilization with methanol. The cells were then stained with CD33 PE, CD20 PerCP‐Cy5.5, CD4 PE‐Cy7, CD3 Ax700, pStat1 Ax647, and pStat3 Ax488 and run on an LSR2 cytometer (with digital acquisition). In step 1 (A), debris and red blood cells are first eliminated based on scatter characteristics, followed by gating of monocytes and B cells based on expression of CD20 and CD33. The double negative cells are further gated into CD4+ and CD4 T cells based on expression of CD3 and CD4. In step 2 (B), the response of these four cell types to IFN‐γ and IL‐10 stimulation is analyzed. Stat1 is phosphorylated in response to IFN‐γ stimulation only in monocytes and B cells. Stat3 is phosphorylated in all cell types, but only in response to IL‐10 stimulation.

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