Single‐Cell Phospho‐Protein Analysis by Flow Cytometry

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 to take standard biochemical analysis into primary cells to garner valuable information about signaling events in physiologic settings. Curr. Protoc. Immunol. 78:8.17.1‐8.17.20. © 2007 by John Wiley & Sons, Inc.