University of Worcester Worcester Research and Publications
 
  USER PANEL:
  ABOUT THE COLLECTION:
  CONTACT DETAILS:

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

Wadley, Alex, Morgan, R.G., Heesom, K.J., Hole, P.S. and Coles, Steven ORCID: https://orcid.org/0000-0002-1109-6971 (2018) Detecting Intracellular Thiol Redox State in Leukaemia and Heterogeneous Immune Cell Populations: an Optimised Protocol for Digital Flow Cytometers. MethodsX, 5. pp. 1473-1483. ISSN 2215-0161

[img] Text
Wadley_et_al_2018_MethodsX_Final_Version.docx
Restricted to Repository staff only

Download (79kB) | Request a copy
[img]
Preview
Text
Wadley_et_al_2018_MethodsX_Final_Version.pdf - Accepted Version
Available under License Creative Commons Attribution.

Download (158kB) | Preview

Abstract

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].

Item Type: Article
Additional Information:

The full-text of the published version can be accessed via the Official URL.

Uncontrolled Discrete Keywords: flow cytometry, reactive oxygen species, oxidative stress, fluorescein-5 maleimide, reductive stress
Subjects: Q Science > QD Chemistry
Q Science > QH Natural history > QH301 Biology
Q Science > QR Microbiology > QR180 Immunology
R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Divisions: College of Health, Life and Environmental Sciences > School of Science and the Environment
Related URLs:
Copyright Info: Open Access article (UW LS APC)
Depositing User: Steven Coles
Date Deposited: 26 Oct 2018 08:50
Last Modified: 17 Jun 2020 17:25
URI: https://eprints.worc.ac.uk/id/eprint/7168

Actions (login required)

View Item View Item
 
     
Worcester Research and Publications is powered by EPrints 3 which is developed by the School of Electronics and Computer Science at the University of Southampton. More information and software credits.