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Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes

Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH(2)-DA) as fluorogenic redox probe is problematic in liver cell lines because of membrane transport proteins that interfere with probe kinetics, among other...

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Autores principales: Reiniers, Megan J., de Haan, Lianne R., Reeskamp, Laurens F., Broekgaarden, Mans, van Golen, Rowan F., Heger, Michal
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8147027/
https://www.ncbi.nlm.nih.gov/pubmed/33925917
http://dx.doi.org/10.3390/antiox10050674
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author Reiniers, Megan J.
de Haan, Lianne R.
Reeskamp, Laurens F.
Broekgaarden, Mans
van Golen, Rowan F.
Heger, Michal
author_facet Reiniers, Megan J.
de Haan, Lianne R.
Reeskamp, Laurens F.
Broekgaarden, Mans
van Golen, Rowan F.
Heger, Michal
author_sort Reiniers, Megan J.
collection PubMed
description Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH(2)-DA) as fluorogenic redox probe is problematic in liver cell lines because of membrane transport proteins that interfere with probe kinetics, among other reasons. The properties of DCFH(2)-DA were analyzed in hepatocytes (HepG2, HepaRG) to characterize methodological issues that could hamper data interpretation and falsely skew conclusions. Experiments were focused on probe stability in relevant media, cellular probe uptake/retention/excretion, and basal oxidant formation and metabolism. DCFH(2)-DA was used under optimized experimental conditions to intravitally visualize and quantify oxidative stress in real-time in HepG2 cells subjected to anoxia/reoxygenation. The most important findings were that: (1) the non-fluorescent DCFH(2)-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH(2) oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH(2)-DA in cell culture medium relied on medium composition. The use of DCFH(2)-DA to measure oxidative stress in cultured hepatocytes comes with methodological and technical challenges, which were characterized and solved. Optimized in vitro and intravital imaging protocols were formulated to help researchers conduct proper experiments and draw robust conclusions.
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spelling pubmed-81470272021-05-26 Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes Reiniers, Megan J. de Haan, Lianne R. Reeskamp, Laurens F. Broekgaarden, Mans van Golen, Rowan F. Heger, Michal Antioxidants (Basel) Article Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH(2)-DA) as fluorogenic redox probe is problematic in liver cell lines because of membrane transport proteins that interfere with probe kinetics, among other reasons. The properties of DCFH(2)-DA were analyzed in hepatocytes (HepG2, HepaRG) to characterize methodological issues that could hamper data interpretation and falsely skew conclusions. Experiments were focused on probe stability in relevant media, cellular probe uptake/retention/excretion, and basal oxidant formation and metabolism. DCFH(2)-DA was used under optimized experimental conditions to intravitally visualize and quantify oxidative stress in real-time in HepG2 cells subjected to anoxia/reoxygenation. The most important findings were that: (1) the non-fluorescent DCFH(2)-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH(2) oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH(2)-DA in cell culture medium relied on medium composition. The use of DCFH(2)-DA to measure oxidative stress in cultured hepatocytes comes with methodological and technical challenges, which were characterized and solved. Optimized in vitro and intravital imaging protocols were formulated to help researchers conduct proper experiments and draw robust conclusions. MDPI 2021-04-26 /pmc/articles/PMC8147027/ /pubmed/33925917 http://dx.doi.org/10.3390/antiox10050674 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Reiniers, Megan J.
de Haan, Lianne R.
Reeskamp, Laurens F.
Broekgaarden, Mans
van Golen, Rowan F.
Heger, Michal
Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes
title Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes
title_full Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes
title_fullStr Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes
title_full_unstemmed Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes
title_short Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes
title_sort analysis and optimization of conditions for the use of 2′,7′-dichlorofluorescein diacetate in cultured hepatocytes
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8147027/
https://www.ncbi.nlm.nih.gov/pubmed/33925917
http://dx.doi.org/10.3390/antiox10050674
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