Detecting intracellular thiol redox state in leukaemia and heterogeneous immune cell populations: an optimised protocol for digital flow cytometers

Wadley, Alex J, Morgan, Rhys, Heesom, Kate J, Hole, Paul S and Coles, Steven J (2018) Detecting intracellular thiol redox state in leukaemia and heterogeneous immune cell populations: an optimised protocol for digital flow cytometers. Methods X, 5. pp. 1473-1483. ISSN 2215-0161

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Abstract

Flow cytometric methods for detecting and quantifying reduced intracellular thiol content using fluorescein-5-maleimide (F5M) in viable eukaryotic cells date back to 1983 [1]. There has been little development in these methodologies since that time, a period that has witnessed huge technological advances, particularly with the emergence of digital multi-parameter flow cytometric systems. Concurrent advancement in our understanding of redox regulation within eukaryotic cellular systems has also followed, whereby it is now accepted that cysteine thiols partake in redox reactions, which regulate protein activity and function [2,3]. Moreover, we are at the dawn of a new era in redox biology whereby the importance of ‘reductive stress’ in eukaryotic cellular systems is gathering momentum [4]. It is therefore critical that methods be continually advanced to better understand these concepts in more detail at the cellular level. Flow cytometry is a powerful technique that may be used for this purpose. Henceforth we have rejuvenated these methods to address modern scientific questions. In this paper, essential detail is provided on:

• The adaption of a protocol initially described by Durand & Olive [1] for use with modern digital flow cytometer configurations. Here we provide optimal conditions for labelling intracellular thiols with F5M for detection using digital flow cytometers. Our modifications avoid the use of methanol fixation thus preserving cell viability in single cell suspension cultures.

• Demonstration that flow cytometry can detect the gain and loss of reduced intracellular thiols in cells exposed to physiological doses of hydrogen peroxide mediated by glucose oxidase [5].

• Validation of F5M protein labelling by coupling method to confocal microscopy and downstream proteomics, thus permitting a powerful experimental platform for potential use with next generation flow cytometry e.g. CyTOF [6].

Item Type: Article
Schools and Departments: School of Life Sciences > Biochemistry
Research Centres and Groups: Haematology Research Group
Subjects: Q Science > Q Science (General) > Q0179.9 Research
Related URLs:
Depositing User: Rhys Morgan
Date Deposited: 20 Nov 2018 10:03
Last Modified: 11 Jul 2019 14:30
URI: http://sro.sussex.ac.uk/id/eprint/79541

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