UNIT 8.17 Single-Cell Phospho-Protein Analysis by Flow Cytometry

  1. Kenneth R. Schulz,
  2. Erika A. Danna,
  3. Peter O. Krutzik,
  4. Garry P. Nolan

Published Online: 1 FEB 2012

DOI: 10.1002/0471142735.im0817s96

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Schulz, K. R., Danna, E. A., Krutzik, P. O. and Nolan, G. P. 2012. Single-Cell Phospho-Protein Analysis by Flow Cytometry. Current Protocols in Immunology. 96:IV:8.17:8.17.1–8.17.20.

Author Information

  1. Stanford University, Stanford, California

Publication History

  1. Published Online: 1 FEB 2012
  2. Published Print: FEB 2012


This protocol describes methods for monitoring intracellular phosphorylation-dependent signaling events on a single-cell basis. This approach measures cell signaling by treating cells with exogenous stimuli, fixing cells with formaldehyde, permeabilizing with methanol, and then staining with phospho-specific antibodies. Thus, cell signaling states can be determined as a measure of how cells interact with their environment. This method has applications in clinical research as well as mechanistic studies of basic biology. In clinical research, diagnostic or drug efficacy information can be retrieved by discovering how a disease affects the ability of cells to respond to growth factors. Basic scientists can use this technique to analyze signaling events in cell lines and human or murine primary cells, including rare populations, like B1 cells or stem cells. This technique has broad applications bringing standard biochemical analysis into primary cells in order to garner valuable information about signaling events in physiologic settings. Curr. Protoc. Immunol. 96:8.17.1-8.17.20. © 2012 by John Wiley & Sons, Inc.


  • phosphorylation;
  • signaling;
  • flow cytometry;
  • FACS;
  • intracellular staining