Experimental Conditions That Influence the Utility of 2'7'-Dichlorodihydrofluorescein Diacetate (DCFH2-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

doi:10.3390/antiox11081424

Experimental Conditions That Influence the Utility of 2'7'-Dichlorodihydrofluorescein Diacetate (DCFH₂-DA) as a Fluorogenic Biosensor for Mitochondrial Redox Status

Antioxidants (Basel). 2022 Jul 22;11(8):1424. doi: 10.3390/antiox11081424.

Authors

Lianne R de Haan^{1

2}, Megan J Reiniers^{1

3

4}, Laurens F Reeskamp⁵, Ali Belkouz⁶, Lei Ao¹, Shuqun Cheng⁷, Baoyue Ding¹, Rowan F van Golen⁸, Michal Heger^{1

2

4

9}

Affiliations

¹ Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics, Department of Pharmaceutics, College of Medicine, Jiaxing University, Jiaxing 314001, China.
² Laboratory for Experimental Oncology, Department of Pathology, Erasmus MC, 3015 GD Rotterdam, The Netherlands.
³ Department of Surgery, Haaglanden Medisch Centrum, 2262 BA The Hague, The Netherlands.
⁴ Membrane Biochemistry and Biophysics, Department of Chemistry, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands.
⁵ Department of Vascular Medicine, Amsterdam University Medical Centers, Location AMC, 1105 AZ Amsterdam, The Netherlands.
⁶ Department of Medical Oncology, Amsterdam University Medical Centers, Location AMC, University of Amsterdam, Cancer Center Amsterdam, 1105 AZ Amsterdam, The Netherlands.
⁷ Department of Hepatic Surgery VI, The Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai 200438, China.
⁸ Department of Gastroenterology and Hepatology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands.
⁹ Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CG Utrecht, The Netherlands.

Abstract

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.

Keywords: antimycin A; electron transport chain inhibitors; fluorescence imaging; fluorogenic redox probe; hepatocytes; myxothiazol; oxidative stress; piericidin A; rotenone.

Abstract

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