4.7 Article

Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications

Journal

CLINICAL IMMUNOLOGY
Volume 110, Issue 3, Pages 206-221

Publisher

ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.clim.2003.11.009

Keywords

intracellular; phosphorylation; multiparameter; phospho-specific antibodies; activation; drug screening; disease characterization

Categories

Funding

  1. NHLBI NIH HHS [N01-HV-28183I] Funding Source: Medline

Ask authors/readers for more resources

Analysis of protein phosphorylation with flow cytometric techniques has emerged as a powerful tool in the field of immunological signaling, allowing cellular subsets in complex populations to be analyzed accurately and rapidly. In this review, we examine the development of phospho-epitope, or phospho-specific, flow cytometry and the premises upon which the technique is based. Phospho-specific flow cytometry is compared to traditional biochemical methods, and its advantages, such as single cell analysis, multiparameter data acquisition, rapid protocols, and the ability to analyze rare cell subsets, are detailed. We also discuss the many technical considerations that must be addressed when developing new antibodies or analyzing new epitopes including antigen accessibility, stability of the phosphoepitope, fluorophore selection, surface phenotype integrity, and antibody suitability for staining epitopes inside fixed and permeabilized cells. The methods that have been used to date are described in light of these technical considerations. The importance of developing bioinformatic platforms in parallel with these techniques is emphasized due to the large, multiparameter datasets that are rapidly accumulated and which require more efficient data viewing and complex clustering methods than currently available for flow cytometric data. Finally, we discuss the potential clinical applications of phospho-specific flow cytometry in analyzing immune cell development and antigen-specific immune responses, as well as pharmacodynamic profiling of disease states or drug efficacy and specificity against particular signaling proteins. (C) 2004 Elsevier Inc. All rights reserved.

Authors

I am an author on this paper
Click your name to claim this paper and add it to your profile.

Reviews

Primary Rating

4.7
Not enough ratings

Secondary Ratings

Novelty
-
Significance
-
Scientific rigor
-
Rate this paper

Recommended

No Data Available
No Data Available