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

Research output: Contribution to journalArticlepeer-review

Standard

Detecting intracellular thiol redox state in leukaemia and heterogeneous immune cell populations : an optimised protocol for digital flow cytometers. / Wadley, Alex; Morgan, Rhys G.; Heesom, Kate J. ; Hole, Paul S.; Coles, Steven J.

In: MethodsX, Vol. 5, 01.11.2018, p. 1473-1483.

Research output: Contribution to journalArticlepeer-review

Harvard

APA

Vancouver

Author

Bibtex

@article{af2b8ca14d5d4ca2a531371b74fe21db,
title = "Detecting intracellular thiol redox state in leukaemia and heterogeneous immune cell populations: an optimised protocol for digital flow cytometers",
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 (Durand and Olive [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 (Groitl and Jakob (2014), Won et al. (2012)). Moreover, we are at the dawn of a new era in redox biology whereby the importance of {\textquoteleft}reductive stress{\textquoteright} in eukaryotic cellular systems is gathering momentum (Wadley et al. (2018) [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 and 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 (Hole et al. (2013) [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 (Lin and Maecker (2018) [6]).",
author = "Alex Wadley and Morgan, {Rhys G.} and Heesom, {Kate J.} and Hole, {Paul S.} and Coles, {Steven J.}",
year = "2018",
month = nov,
day = "1",
doi = "10.1016/j.mex.2018.10.013",
language = "English",
volume = "5",
pages = "1473--1483",
journal = "MethodsX",
issn = "2215-0161",
publisher = "Elsevier",

}

RIS

TY - JOUR

T1 - Detecting intracellular thiol redox state in leukaemia and heterogeneous immune cell populations

T2 - an optimised protocol for digital flow cytometers

AU - Wadley, Alex

AU - Morgan, Rhys G.

AU - Heesom, Kate J.

AU - Hole, Paul S.

AU - Coles, Steven J.

PY - 2018/11/1

Y1 - 2018/11/1

N2 - Flow cytometric methods for detecting and quantifying reduced intracellular thiol content using fluorescein-5-maleimide (F5M) in viable eukaryotic cells date back to 1983 (Durand and Olive [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 (Groitl and Jakob (2014), Won et al. (2012)). 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 (Wadley et al. (2018) [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 and 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 (Hole et al. (2013) [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 (Lin and Maecker (2018) [6]).

AB - Flow cytometric methods for detecting and quantifying reduced intracellular thiol content using fluorescein-5-maleimide (F5M) in viable eukaryotic cells date back to 1983 (Durand and Olive [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 (Groitl and Jakob (2014), Won et al. (2012)). 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 (Wadley et al. (2018) [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 and 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 (Hole et al. (2013) [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 (Lin and Maecker (2018) [6]).

UR - http://europepmc.org/articles/PMC6251017

U2 - 10.1016/j.mex.2018.10.013

DO - 10.1016/j.mex.2018.10.013

M3 - Article

C2 - 30505701

VL - 5

SP - 1473

EP - 1483

JO - MethodsX

JF - MethodsX

SN - 2215-0161

ER -