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Using flow cytometry to detect and measure intracellular thiol redox status in viable T cells from heterogeneous populations

Wadley, Alex J., Morgan, Rhys G., Darley, Richard L., Hole, Paul S. and Coles, Steven J. 2019. Using flow cytometry to detect and measure intracellular thiol redox status in viable T cells from heterogeneous populations. In: Hancock, John T. and Conway, Myra E. eds. Redox-Mediated Signal Transduction: Methods and Protocols, Vol. 1990. Methods in Molecular Biology, New York, NY, USA: Humana Press, pp. 53-70. (10.1007/978-1-4939-9463-2_5)

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Abstract

Increased production of reactive oxygen species (ROS) and deficiencies in cellular antioxidant defenses are the principal causes of cellular oxidative stress. ROS can react with a variety intracellular molecules, including redox active cysteine thiols (–SH) within proteins. Cysteine thiols can occupy several redox states and conversion between them is highly dynamic during, for example, cell growth, resulting in modification and subsequent loss of the “reduced thiol” form (–SH or –S−). The challenge lies with detecting and measuring thiol redox status inside viable heterogeneous cell populations (e.g., peripheral blood mononuclear cells (PBMCs)). Here we describe a flow cytometric approach for the evaluation of intracellular thiol redox status in human CD3+ T cells within a viable PBMC preparation. Using the thiol reactive probe, fluorescein-5 maleimide (F5M), we demonstrate that loss of reduced intracellular thiol correlates with a decrease in F5M fluorescence. We also detected a loss of F5M fluorescence in Jurkat cell cultures exposed to exogenous H2O2 generated by glucose oxidase. Since F5M binds irreversibly to reduced cysteine thiols, cells may be sorted based on F5M fluorescence intensity and redox active proteins can subsequently be extracted and separated using SDS-PAGE. This final step facilitates identification of redox active proteins from individual cell populations in live heterogeneous cell mixes using proteomic analysis.

Item Type: Book Section
Date Type: Published Online
Status: Published
Schools: Medicine
Publisher: Humana Press
ISBN: 9781493994618
ISSN: 1064-3745
Date of First Compliant Deposit: 29 May 2020
Date of Acceptance: 31 May 2019
Last Modified: 01 Jun 2020 11:00
URI: http://orca.cardiff.ac.uk/id/eprint/131975

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