Optimal Use of 2',7'-Dichlorofluorescein Diacetate in Cultured Hepatocytes

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 (DCFH₂-DA) is one of the most widely used fluorogenic probes for the detection of reactive transients. The nonfluorescent DCFH₂-DA crosses the plasma membrane and is deacetylated by cytosolic esterases to 2',7'-dichlorodihydrofluorescein (DCFH₂). The nonfluorescent DCFH₂ is subsequently oxidized by reactive transients to form the fluorescent 2',7'-dichlorofluorescein (DCF). The use of DCFH₂-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 DCFH₂-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 DCFH₂-DA and DCF are taken up rapidly, (2) DCF is poorly retained in the cytosol and exits the cell, (3) the rate of DCFH₂ oxidation is cell type-specific, (4) DCF fluorescence intensity is pH-dependent at pH < 7, and (5) the stability of DCFH₂-DA in cell culture medium relies on medium composition. Based on the findings, the conditions for the use of DCFH₂-DA in hepatocyte cell lines were optimized. Finally, the optimized protocol was reduced to practice and DCFH₂-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.