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

Abstract

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 

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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

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