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<sub>2</sub>-DA) as fluorogenic redox probe is problematic in liver cell lines because of membrane transport proteins that interfere with probe...

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Main Authors:	Megan J. Reiniers, Lianne R. de Haan, Laurens F. Reeskamp, Mans Broekgaarden, Rowan F. van Golen, Michal Heger
Format:	Article
Language:	English
Published:	MDPI AG 2021-04-01
Series:	Antioxidants
Subjects:	fluorogenic redox probe oxidative and nitrosative stress liver diseases hepatocytes cellular uptake and export intravital fluorescence imaging
Online Access:	https://www.mdpi.com/2076-3921/10/5/674

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spelling	doaj-0ac759543ed44582b1bf42a92f2e21b12021-04-26T23:02:21ZengMDPI AGAntioxidants2076-39212021-04-011067467410.3390/antiox10050674Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured HepatocytesMegan J. Reiniers0Lianne R. de Haan1Laurens F. Reeskamp2Mans Broekgaarden3Rowan F. van Golen4Michal Heger5Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics, Department of Pharmaceutics, College of Medicine, Jiaxing University, Jiaxing 314001, ChinaJiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics, Department of Pharmaceutics, College of Medicine, Jiaxing University, Jiaxing 314001, ChinaDepartment of Vascular Medicine, Amsterdam UMC, Location AMC, 1105 AZ Amsterdam, The NetherlandsTeam Cancer Targets and Experimental Therapeutics, Department Microenvironment Cell Plasticity and Signaling, Institute for Advanced Biosciences, CNRS UMR 5309, Université de Grenoble-Alpes, Allée des Alpes, 38700 La Tronche, FranceDepartment of Gastroenterology and Hepatology, Leiden University Medical Center, 2333 ZA Leiden, The NetherlandsJiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics, Department of Pharmaceutics, College of Medicine, Jiaxing University, Jiaxing 314001, ChinaNumerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH<sub>2</sub>-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<sub>2</sub>-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<sub>2</sub>-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<sub>2</sub>-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH<sub>2</sub> oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH<sub>2</sub>-DA in cell culture medium relied on medium composition. The use of DCFH<sub>2</sub>-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.https://www.mdpi.com/2076-3921/10/5/674fluorogenic redox probeoxidative and nitrosative stressliver diseaseshepatocytescellular uptake and exportintravital fluorescence imaging
collection	DOAJ
language	English
format	Article
sources	DOAJ
author	Megan J. Reiniers Lianne R. de Haan Laurens F. Reeskamp Mans Broekgaarden Rowan F. van Golen Michal Heger
spellingShingle	Megan J. Reiniers Lianne R. de Haan Laurens F. Reeskamp Mans Broekgaarden Rowan F. van Golen Michal Heger Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes Antioxidants fluorogenic redox probe oxidative and nitrosative stress liver diseases hepatocytes cellular uptake and export intravital fluorescence imaging
author_facet	Megan J. Reiniers Lianne R. de Haan Laurens F. Reeskamp Mans Broekgaarden Rowan F. van Golen Michal Heger
author_sort	Megan J. Reiniers
title	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_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_sort	analysis and optimization of conditions for the use of 2′,7′-dichlorofluorescein diacetate in cultured hepatocytes
publisher	MDPI AG
series	Antioxidants
issn	2076-3921
publishDate	2021-04-01
description	Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH<sub>2</sub>-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<sub>2</sub>-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<sub>2</sub>-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<sub>2</sub>-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH<sub>2</sub> oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH<sub>2</sub>-DA in cell culture medium relied on medium composition. The use of DCFH<sub>2</sub>-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.
topic	fluorogenic redox probe oxidative and nitrosative stress liver diseases hepatocytes cellular uptake and export intravital fluorescence imaging
url	https://www.mdpi.com/2076-3921/10/5/674
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Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes

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