Detecting Intracellular Thiol Redox State in Leukaemia and Heterogeneous Immune Cell Populations: an Optimised Protocol for Digital Flow Cytometers

Flow cytometric methods for detecting and quantifying reduced intracellular thiol content using fluorescein-5-maleimide 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.
• 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].