Unit

UNIT 6.20 Multiparameter Analysis of Intracellular Phosphoepitopes in Immunophenotyped Cell Populations by Flow Cytometry

  1. Omar D. Perez1,
  2. Dennis Mitchell1,
  3. Roberto Campos2,
  4. Guo-Jian Gao2,
  5. Li Li2,
  6. Garry P. Nolan1

Published Online: 1 MAY 2005

DOI: 10.1002/0471142956.cy0620s32

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Perez, O. D., Mitchell, D., Campos, R., Gao, G.-J., Li, L. and Nolan, G. P. 2005. Multiparameter Analysis of Intracellular Phosphoepitopes in Immunophenotyped Cell Populations by Flow Cytometry. Current Protocols in Cytometry. 32:6.20:6.20.1–6.20.22.

Author Information

  1. 1

    Stanford University School of Medicine, Stanford, California

  2. 2

    BD Biosciences Pharmingen, San Diego, California

Publication History

  1. Published Online: 1 MAY 2005
  2. Published Print: APR 2005

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