Experimental Conditions That Influence the Utility of 2′7′-Dichlorodihydrofluorescein Diacetate (DCFH<sub>2</sub>-DA) as a Fluorogenic Biosensor for Mitochondrial Redox Status
Lianne R. de Haan,
Megan J. Reiniers,
Laurens F. Reeskamp,
Ali Belkouz,
Lei Ao,
Shuqun Cheng,
Baoyue Ding,
Rowan F. van Golen,
Michal Heger
Affiliations
Lianne R. de Haan
Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics, Department of Pharmaceutics, College of Medicine, Jiaxing University, Jiaxing 314001, China
Megan J. Reiniers
Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics, Department of Pharmaceutics, College of Medicine, Jiaxing University, Jiaxing 314001, China
Laurens F. Reeskamp
Department of Vascular Medicine, Amsterdam University Medical Centers, Location AMC, 1105 AZ Amsterdam, The Netherlands
Ali Belkouz
Department of Medical Oncology, Amsterdam University Medical Centers, Location AMC, University of Amsterdam, Cancer Center Amsterdam, 1105 AZ Amsterdam, The Netherlands
Lei Ao
Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics, Department of Pharmaceutics, College of Medicine, Jiaxing University, Jiaxing 314001, China
Shuqun Cheng
Department of Hepatic Surgery VI, The Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai 200438, China
Baoyue Ding
Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics, Department of Pharmaceutics, College of Medicine, Jiaxing University, Jiaxing 314001, China
Rowan F. van Golen
Department of Gastroenterology and Hepatology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
Michal Heger
Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics, Department of Pharmaceutics, College of Medicine, Jiaxing University, Jiaxing 314001, China
Oxidative stress has been causally linked to various diseases. Electron transport chain (ETC) inhibitors such as rotenone and antimycin A are frequently used in model systems to study oxidative stress. Oxidative stress that is provoked by ETC inhibitors can be visualized using the fluorogenic probe 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH2-DA). Non-fluorescent DCFH2-DA crosses the plasma membrane, is deacetylated to 2′,7′-dichlorodihydrofluorescein (DCFH2) by esterases, and is oxidized to its fluorescent form 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence can, therefore, be used as a semi-quantitative measure of general oxidative stress. However, the use of DCFH2-DA is complicated by various protocol-related factors that mediate DCFH2-to-DCF conversion independently of the degree of oxidative stress. This study therefore analyzed the influence of ancillary factors on DCF formation in the context of ETC inhibitors. It was found that ETC inhibitors trigger DCF formation in cell-free experiments when they are co-dissolved with DCFH2-DA. Moreover, the extent of DCF formation depended on the type of culture medium that was used, the pH of the assay system, the presence of fetal calf serum, and the final DCFH2-DA solvent concentration. Conclusively, experiments with DCFH2-DA should not discount the influence of protocol-related factors such as medium and mitochondrial inhibitors (and possibly other compounds) on the DCFH2-DA-DCF reaction and proper controls should always be built into the assay protocol.
