Protocol

Flow Cytometry Protocols

Volume 263 of the series Methods in Molecular Biology™ pp 67-94

Flow Cytometric Analysis of Kinase Signaling Cascades

  • Omar D. PerezAffiliated withThe Baxter Laboratory for Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University School of Medicine
  • , Peter O. KrutzikAffiliated withThe Baxter Laboratory for Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University School of Medicine
  • , Garry P. NolanAffiliated withThe Baxter Laboratory for Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University School of Medicine

* Final gross prices may vary according to local VAT.

Get Access

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