Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics, Department of Pharmaceutics, College of Medicine, Jiaxing University, Jiaxing, Zhejiang, P. R. China.
Department of Surgery, Haaglanden Medisch Centrum, The Hague, The Netherlands.
Methods Mol Biol. 2022;2451:721-747. doi: 10.1007/978-1-0716-2099-1_39.
Oxidative stress is a state that arises when the production of reactive transients overwhelms the cell's capacity to neutralize the oxidants and radicals. This state often coincides with the pathogenesis and perpetuation of numerous chronic diseases. On the other hand, medical interventions such as radiation therapy and photodynamic therapy generate radicals to selectively damage and kill diseased tissue. As a result, the qualification and quantification of oxidative stress are of great interest to those studying disease mechanisms as well as therapeutic interventions. 2',7'-Dichlorodihydrofluorescein-diacetate (DCFH-DA) is one of the most widely used fluorogenic probes for the detection of reactive transients. The nonfluorescent DCFH-DA crosses the plasma membrane and is deacetylated by cytosolic esterases to 2',7'-dichlorodihydrofluorescein (DCFH). The nonfluorescent DCFH is subsequently oxidized by reactive transients to form the fluorescent 2',7'-dichlorofluorescein (DCF). The use of DCFH-DA in hepatocyte-derived cell lines is more challenging because of membrane transport proteins that interfere with probe uptake and retention, among several other reasons. Cancer cells share some of the physiological and biochemical features with hepatocytes, so probe-related technical issues are applicable to cultured malignant cells as well. This study therefore analyzed the in vitro properties of DCFH-DA in cultured human hepatocytes (HepG2 cells and differentiated and undifferentiated HepaRG cells) to identify methodological and technical features that could impair proper data analysis and interpretation. The main issues that were found and should therefore be accounted for in experimental design include the following: (1) both DCFH-DA and DCF are taken up rapidly, (2) DCF is poorly retained in the cytosol and exits the cell, (3) the rate of DCFH oxidation is cell type-specific, (4) DCF fluorescence intensity is pH-dependent at pH < 7, and (5) the stability of DCFH-DA in cell culture medium relies on medium composition. Based on the findings, the conditions for the use of DCFH-DA in hepatocyte cell lines were optimized. Finally, the optimized protocol was reduced to practice and DCFH-DA was applied to visualize and quantify oxidative stress in real time in HepG2 cells subjected to anoxia/reoxygenation as a source of reactive transients.
氧化应激是一种当活性瞬变的产生超过细胞中和氧化剂和自由基的能力时出现的状态。这种状态通常与许多慢性疾病的发病机制和持续存在有关。另一方面，放射治疗和光动力疗法等医学干预会产生自由基，以选择性地破坏和杀死患病组织。因此，氧化应激的定性和定量对于研究疾病机制和治疗干预的人来说非常感兴趣。2',7'-二氯二氢荧光素二乙酸酯 (DCFH-DA) 是用于检测活性瞬变的最广泛使用的荧光探针之一。非荧光的 DCFH-DA 通过质膜，并被细胞质酯酶去乙酰化为 2',7'-二氯二氢荧光素 (DCFH)。随后，非荧光的 DCFH 被活性瞬态氧化形成荧光的 2',7'-二氯荧光素 (DCF)。由于膜转运蛋白干扰探针摄取和保留等原因，在肝细胞衍生的细胞系中使用 DCFH-DA 更具挑战性。由于癌症细胞与肝细胞具有一些生理和生化特征，因此与探针相关的技术问题也适用于培养的恶性细胞。因此，本研究分析了培养的人肝细胞（HepG2 细胞和分化和未分化的 HepaRG 细胞）中 DCFH-DA 的体外特性，以确定可能会影响正确数据分析和解释的方法学和技术特征。发现并因此在实验设计中应考虑的主要问题包括以下几点：(1) DCFH-DA 和 DCF 都被迅速摄取，(2) DCF 在细胞质中保留不佳并离开细胞，(3) DCFH 氧化速率是细胞类型特异性的，(4) 在 pH < 7 时，DCF 荧光强度依赖于 pH，(5) DCFH-DA 在细胞培养基中的稳定性取决于培养基成分。基于这些发现，优化了用于肝细胞系的 DCFH-DA 的使用条件。最后，将优化的方案付诸实践，并将 DCFH-DA 应用于实时可视化和定量缺氧/复氧处理的 HepG2 细胞中的氧化应激，作为活性瞬变的来源。
