Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes
Abstract
:1. Introduction
2. Methods
2.1. Reagents and Buffers
2.2. Preparation of DCFH2
2.3. Determination of Molar Extinction Coefficients
2.4. Spectral Properties of DCFH2-DA, DCFH2, and DCF
2.5. Stability of DCFH2-DA and DCFH2 in Solvent
2.6. Cell Culture
2.7. Cellular DCFH2-DA Uptake
2.8. Cellular DCF Uptake
2.9. Intracellular DCF Retention and Transmembrane Diffusion
2.10. Basal Oxidant Formation and Cellular Metabolic Rate
2.11. Real-Time Analysis of Oxidant Formation during In Vitro Anoxia/Reoxygenation in HepG2 Cells
2.12. Statistical Analysis
3. Results
3.1. The Spectral Properties of DCFH2-DA and Derivatives Are pH-Dependent
3.2. The Stability of DCFH2-DA and DCFH2 in Aqueous Solvent and Medium Is Dependent on the Composition of the Solution
3.3. DCFH2-DA Rapidly Accumulates in HepG2 and HepaRG Cells
3.4. DCF Accumulates in HepG2 and HepaRG Cells and Is Poorly Retained
3.5. DCF Crosses Membranes
3.6. Basal Oxidant Formation and Cellular Metabolic Rate Differ between HepG2 and HepaRG Cells
3.7. Oxidative Stress during In Vitro Anoxia/Reoxygenation Can Be Visualized in Real-Time Using DCFH2-DA
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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DMEM | RPMI | WE | ||
---|---|---|---|---|
Salts | Fe3+ (nitrate) | 0.25 | 0.00 | 0.00 |
Vitamins | Ascorbic acid | 0.00 | 0.00 | 11.36 |
Riboflavin | 1.06 | 0.53 | 0.27 | |
Amino acids | Cysteine | 0.00 | 0.00 | 330.17 |
Histidine | 270.69 | 96.67 | 96.67 | |
Methionine | 201.06 | 100.53 | 100.53 | |
Phenylalanine | 399.54 | 90.80 | 151.34 | |
Tryptophan | 78.34 | 24.48 | 48.96 | |
Tyrosine | 397.37 | 110.38 | 193.17 | |
Other compounds | GSH | 0.00 | 3.25 | 0.16 |
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Reiniers, M.J.; de Haan, L.R.; Reeskamp, L.F.; Broekgaarden, M.; van Golen, R.F.; Heger, M. Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes. Antioxidants 2021, 10, 674. https://doi.org/10.3390/antiox10050674
Reiniers MJ, de Haan LR, Reeskamp LF, Broekgaarden M, van Golen RF, Heger M. Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes. Antioxidants. 2021; 10(5):674. https://doi.org/10.3390/antiox10050674
Chicago/Turabian StyleReiniers, Megan J., Lianne R. de Haan, Laurens F. Reeskamp, Mans Broekgaarden, Rowan F. van Golen, and Michal Heger. 2021. "Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes" Antioxidants 10, no. 5: 674. https://doi.org/10.3390/antiox10050674