Multiparameter Analysis of Intracellular Phosphoepitopes in Immunophenotyped Cell Populations by Flow Cytometry

Omar D. Perez1, Dennis Mitchell1, Roberto Campos2, Guo‐Jian Gao2, Li Li2, Garry P. Nolan1

1 Stanford University School of Medicine, Stanford, California, 2 BD Biosciences Pharmingen, San Diego, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.20
DOI:  10.1002/0471142956.cy0620s32
Online Posting Date:  May, 2005
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Abstract

This unit presents protocols for measuring intracellular phosphoepitopes by flow cytometry for biochemical investigations in both human and murine primary cells as well as in cell lines. Conventional methods that require cellular lysis cannot discriminate between proteins from different cellular subsets. Intracellular detection of phosphorylated and nonphosphorylated signaling molecules, on the other hand, has recently exposed the heterogeneity that can be observed upon signal transduction. Although staining methodologies for cytokines and cell cycle antigens have been appreciated for years, detection of phosphorylated molecules presents several new challenges, including generation of reagents and details of the staining technique. As these techniques are adapted to new applications, the protocols continue to be refined. This unit describes signal amplification of intracellular signals upon detergent‐based permeabilizations, staining protocol for adherent cells, methodology for whole‐blood staining, and multiparameter staining procedures for surface and intracellular antigens.

Keywords: phosphoproteins; kinase activation; flow cytometry; proteomics; single‐cell

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Flow Cytometric Analysis of Intracellular Phosphoepitopes Using Alcohol‐Based Permeabilization
  • Basic Protocol 2: Flow Cytometric Analysis of Intracellular Phosphoepitopes Using Detergent‐Based Permeabilization
  • Basic Protocol 3: Flow Cytometric Analysis of Intracellular or Surface Phosphoepitopes Using Whole‐Blood Staining
  • Basic Protocol 4: Flow Cytometric Analysis of Cell‐Surface and Intracellular Phosphoepitopes Using Alcohol‐Based Permeabilization
  • Basic Protocol 5: Flow Cytometric Analysis of Cell‐Surface and Intracellular Phosphoepitopes Using Saponin‐Based Permeabilization
  • Alternate Protocol 1: Two‐Step Intracellular and Surface Staining for Flow Cytometric Analysis of Phosphoepitopes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Flow Cytometric Analysis of Intracellular Phosphoepitopes Using Alcohol‐Based Permeabilization

  Materials
  • 1 × 106 cells/condition of stimulation in 1 ml medium
  • 1 × 106 cells/unstimulated condition in 1 ml medium
  • 1 × 106 cells as blank in 1 ml of medium
  • Stimuli of interest
  • 16% paraformaldehyde, methanol‐free (Electron Microscopy Sciences)
  • 90% (v/v) methanol, ice‐cold
  • Staining buffer 1 (see recipe)
  • Pretitered, fluorescently conjugated phosphospecific antibody
  • Antibody capture beads for compensation controls (unit 1.14)
  • 12 × 75–mm polystyrene tubes
  • Refrigerated centrifuge
  • Microscope slides and hemocytometer
  • Flow cytometer with appropriate band‐pass filters

Basic Protocol 2: Flow Cytometric Analysis of Intracellular Phosphoepitopes Using Detergent‐Based Permeabilization

  Materials
  • 1 × 106 cells/condition of stimulation in 1 ml medium
  • 1 × 106 cells/unstimulated condition in 1 ml medium
  • 1 × 106 cells as blank in 1 ml medium
  • 16% paraformaldehyde, methanol‐free (Electron Microscopy Sciences)
  • Phosphate‐buffered saline, pH 7.4 (PBS; appendix 2A)
  • Permeabilization/staining buffer (see recipe)
  • Pretitered, fluorochrome‐conjugated phosphospecific antibody or pretitered unconjugated phosphospecific antibody, anti‐Ig biotinylated antibody, and fluorochrome‐conjugated streptavidin
  • PBS, pH 7.4 ( appendix 2A) containing 1 mM EDTA (store at room temperature)
  • Antibody capture beads for compensation controls (unit 1.14)
  • Refrigerated centrifuge
  • 12 × 75–mm polystyrene tubes
  • Platform shaker
  • Flow cytometer

Basic Protocol 3: Flow Cytometric Analysis of Intracellular or Surface Phosphoepitopes Using Whole‐Blood Staining

  Materials
  • Human donor
  • Activators needed for studying phosphoepitopes of interest
  • 5× BD PhosFlow Whole Blood Lyse/Fix Buffer (BD Biosciences)
  • Hanks' balanced salt solution (HBSS; appendix 2A)
  • 70% (v/v) methanol, cold
  • Staining buffer 2 (see recipe)
  • Pretitered fluorescently conjugated phosphospecific antibody
  • Antibody capture beads for compensation controls (unit 1.14)
  • EDTA‐treated Vacutainers or syringes
  • Centrifuge
  • 12 × 75–mm polystyrene tubes
  • Flow cytometer

Basic Protocol 4: Flow Cytometric Analysis of Cell‐Surface and Intracellular Phosphoepitopes Using Alcohol‐Based Permeabilization

  Materials
  • 1 × 106 cells/condition of stimulation in 1 ml medium
  • 1 × 106 cells/unstimulated condition in 1 ml medium
  • 1 × 106 cells as blank in 1 ml medium
  • Stimuli of interest
  • 16% paraformaldehyde, methanol‐free (Electron Microscopy Sciences)
  • 90% (v/v) methanol, ice‐cold
  • Staining buffer 2 (see recipe)
  • Pretitered, fluorescently conjugated phosphospecific antibody or antibodies
  • Antibody capture beads for compensation controls (unit 1.14)
  • 6‐, 12‐, or 24‐well tissue culture plates
  • 12 × 75–mm polystyrene tubes
  • Refrigerated centrifuge
  • Flow cytometer

Basic Protocol 5: Flow Cytometric Analysis of Cell‐Surface and Intracellular Phosphoepitopes Using Saponin‐Based Permeabilization

  Materials
  • 1 × 106 cells/condition of stimulation in 1 ml medium
  • 1 × 106 cells/unstimulated condition in 1 ml medium
  • 1 × 106 cells as blank in 1 ml medium
  • Stimuli of interest
  • 16% paraformaldehyde, methanol free (Electron Microscopy Sciences)
  • Permeabilization/staining buffer (see recipe)
  • PBS, pH 7.4 ( appendix 2A) containing 1 mM EDTA (store at room temperature)
  • Pretitered, fluorescently conjugated phosphospecific antibody or antibodies
  • Antibody capture beads for compensation controls (unit 1.14)
  • 6‐, 12‐, or 24‐well tissue culture plates
  • 12 × 75–mm tubes
  • Refrigerated centrifuge
  • Platform shaker
  • Flow cytometer

Alternate Protocol 1: Two‐Step Intracellular and Surface Staining for Flow Cytometric Analysis of Phosphoepitopes

  • Phospho wash buffer (see recipe)
  • Extracellular staining buffer (see recipe)
  • Pretitered fluorescently conjugated antibody or antibodies against extracellular phosphoepitopes
  • Fixation buffer: phospho wash buffer (see recipe) containing 1% paraformaldehyde (store at 4°C)
  • Permeabilization buffer: (see recipe)
  • Pretitered fluorescently conjugated antibody or antibodies against intracellular phosphoepitopes
  • Antibody capture beads for compensation controls
  • 96‐well round‐bottom tissue culture plates
  • 96‐well round‐bottom vinyl plates
  • Refrigerated centrifuge with microtiter plate carrier
  • 12 × 75–mm polystyrene tubes
  • Flow cytometer
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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.
   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.
   Hilger, R.A., Kredke, S., Hedley, D., Moeller, J.G., Bauer, R.J., Stellberg, W., Seeber, S., Scheulen, M.E., and Strumberg, D. 2002. ERK1/2 phosphorylation: A biomarker analysis within a phase I study with the new Raf kinase inhibitor BAY43‐9006. Int. J. Clin. Pharmacol. Ther. 40:567‐568.
   Kaech, S.M., Hemby, S., Kersh, E., and Ahmed, R. 2002. Molecular and functional profiling of memory CD8 T cell differentiation. Cell 111:837‐851.
   Krutzik, P.O. and Nolan, G.P. 2003. Intracellular phospho‐protein staining techniques for flow cytometry: Monitoring single cell signaling events. Cytometry 55A:61‐70.
   Krutzik, P.O., Irish, J.M., Nolan, G.P., and Perez, O.D. 2004. Analysis of protein phosphorylation and cellular signaling events by flow cytometry: Techniques and clinical applications. Clin. Immunol. 110:206‐21.
   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., Kinoshita, S., Hitoshi, Y., Payan, D.G., Kitamura, T., Nolan, G.P., and Lorens, J.B. 2002. Activation of the PKB/AKT Pathway by ICAM‐2. Immunity 16:51‐65.
   Perez, O.D., Mitchell, D., Jager, G.C., South, S., Murriel, C., McBride, J., Herzenberg, L.A., Kinoshita, S., and Nolan, G.P. 2003. Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin‐1 and Jun‐activating binding protein 1. Nat. Immunol. 4:1083‐1092.
   Perez, O.D., Krutzik, P.O., and Nolan, G.P. 2004. Flow cytometric analysis of kinase signaling cascades. Methods Mol. Biol. 263:67‐94.
   Rosette, C., Werlen, G., Daniels, M.A., Holman, P.O., Alam, S.M., Travers, P.J., Gascoigne, N.R., Palmer, E., and Jameson, S.C. 2001. The impact of duration versus extent of TCR occupancy on T cell activation: A revision of the kinetic proofreading model. Immunity 15:59‐70.
   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.
   Zell, T. and Jenkins, M.K. 2002. Flow cytometric analysis of T cell receptor signal transduction. Science 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.
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