Single-cell phospho-protein analysis by flow cytometry

Current Protocols in Immunology
Kenneth R SchulzGarry P Nolan

Abstract

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.

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Citations

Jun 1, 2012·Journal of Signal Transduction·Roman M LevytskyyConstantine D Tsoukas
Aug 20, 2011·Journal of Translational Medicine·Michael Kalos
Jun 8, 2012·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Alice M NyakerigaAnjali Joshi
Apr 18, 2014·Journal of Virology·Bailey E FreemanMark K Slifka
Apr 5, 2014·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Cameron A SmurthwaiteRoland Wolkowicz
Jan 6, 2011·Current Protocols in Cytometry·Peter O KrutzikGarry P Nolan
Nov 2, 2018·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Thomas Liechti
Dec 25, 2007·Nature Chemical Biology·Peter O KrutzikGarry P Nolan
Aug 20, 2010·Nature Reviews. Cancer·Walter Kolch, Andrew Pitt
Sep 9, 2010·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Matthew R ClutterGarry P Nolan
Mar 3, 2012·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Bong-Hyun JunDae Hong Jeong
May 23, 2012·The Journal of Immunology : Official Journal of the American Association of Immunologists·Satoshi TanakaFumihiko Ishikawa
Nov 5, 2016·JCI Insight·Yaron CarmiEdgar G Engleman

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