Flow Cytometry Protocols pp 67-94

Part of the Methods in Molecular Biology™ book series (MIMB, volume 263)

Flow Cytometric Analysis of Kinase Signaling Cascades

  • Omar D. Perez
  • Peter O. Krutzik
  • Garry P. Nolan


Flow cytometry offers the capability to assess the heterogeneity of cellular subsets that exist in complex populations, such as peripheral blood, based on immunophenotypes. We describe methodologies to measure phospho-epitopes in single cells as determinants of intracellular kinase activity. Multiparametric staining, using both surface and intracellular stains, allows for the study of discrete biochemical events in readily discernible lymphocyte subsets. As such, the usage of multiparameter flow cytometry to obtain proteomic information provides several major advantages: (1) the ability to perform multiparametric experiments to identify distinct signaling profiles in defined lymphocyte populations, (2) simultaneous correlation of multiple active kinases involved in signaling cascades, (3) profiling of active kinase states to identify signaling signatures of interest rapidly, and (4) biochemical access to rare cell subsets such as those from clinically derived samples or populations that comprise too few in numbers for conventional biochemical analysis.

Key Words

Flow cytometry kinase activation phospho-proteins proteomics single-cell 


  1. 1.
    Perez, O. D. and Nolan, G. P. (2002) Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat. Biotechnol. 20, 155–162.PubMedGoogle Scholar
  2. 2.
    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.PubMedCrossRefGoogle Scholar
  3. 3.
    Perez, O. D., Kinoshita, S., Hitoshi, Y., et al. (2002) Activation of the PKB/AKT pathway by ICAM-2. Immunity 16, 51–65.PubMedCrossRefGoogle Scholar
  4. 4.
    Roederer, M., De Rosa, S., Gerstein, R., et al. (1997) 8 color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity. Cytometry 29, 328–339.PubMedCrossRefGoogle Scholar
  5. 5.
    Baumgarth, N. and Roederer, M. (2000) A practical approach to multicolor flow cytometry for immunophenotyping. J. Immunol. Methods 243, 77–97.PubMedCrossRefGoogle Scholar
  6. 6.
    De Rosa, S. C, Herzenberg, L. A., and Roederer, M. (2001) 11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity. Nat. Med. 7, 245–248.PubMedCrossRefGoogle Scholar
  7. 7.
    Shan, X., Czar, M. J., Bunnell, S. C, et al. (2000) Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyper-responsiveness to CD3 stimulation. Mol. Cell Biol. 20, 6945–6957.PubMedCrossRefGoogle Scholar
  8. 8.
    Freeburn, R. W., Wright, K. L., Burgess, S. J., Astoul, E., Cantrell, D. A., and Ward, S. G. (2002) Evidence that SHIP-1 contributes to phosphatidylinositol 3,4,5-trisphosphate metabolism in T lymphocytes and can regulate novel phosphoinositide 3-kinase effectors. J. Immunol. 169, 5441–5450.PubMedGoogle Scholar
  9. 9.
    Perez, O. D., Mitchell, D., Jager, G. C, et al. (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.PubMedCrossRefGoogle Scholar
  10. 10.
    Roederer, M. (2001) Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 45, 194–205.PubMedCrossRefGoogle Scholar
  11. 11.
    Rudge, E. U., Cutler, A. J., Pritchard, N. R., and Smith, K. G (2002) Interleukin 4 reduces expression of inhibitory receptors on B cells and abolishes CD22 and Fc gamma RII-mediated B cell suppression. J. Exp. Med. 195, 1079–1085.PubMedCrossRefGoogle Scholar
  12. 12.
    Morris, S. C, Dragula, N. L., and Finkelman, F. D. (2002) IL-4 promotes Stat6-dependent survival of autoreactive B cells in vivo without inducing autoantibody production. J. Immunol. 169, 1696–1704.PubMedGoogle Scholar
  13. 13.
    Fiering, S., Northrop, J. P., Nolan, G. P., Mattila, P. S., Crabtree, G. R., and Herzenberg, L. A. (1990) Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor. Genes Dev. 4, 1823–1834.PubMedCrossRefGoogle Scholar
  14. 14.
    Herzenberg, L. A., Parks, D., Sahaf, B., Perez, O., and Roederer, M. (2002) The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin. Client. 48, 1819–1827.Google Scholar
  15. 15.
    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. USA 98, 10,805–10,810.PubMedCrossRefGoogle Scholar
  16. 16.
    Zell, T. and Jenkins, M. K. (2002) Flow cytometric analysis of T cell receptor signal transduction. Science’s STKE, http://stke.sciencemag.org/cgi/content/full/OCsigtrans;2002/128/pl5.
  17. 17.
    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.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Omar D. Perez
    • 1
  • Peter O. Krutzik
    • 1
  • Garry P. Nolan
    • 1
  1. 1.The Baxter Laboratory for Genetic Pharmacology, Department of Microbiology and ImmunologyStanford University School of MedicineStanford

Personalised recommendations