Preparation and Practical Applications of 2′,7′-Dichlorodihydrofluorescein in Redox Assays

Abstract

Oxidative stress, a state in which intra- or extracellular oxidant production outweighs the antioxidative capacity, lies at the basis of many diseases. DCFH ₂-DA (2′,7′-dichlorodihydrofluorescein diacetate) is the most widely used fluorogenic probe for the detection of general oxidative stress. However, the use of DCFH ₂-DA, as many other fluorogenic redox probes, is mainly confined to the detection of intracellular oxidative stress in vitro. To expand the applicability of the probe, an alkaline hydrolysis and solvent extraction procedure was developed to generate high-purity DCFH ₂ (2′,7′-dichlorodihydrofluorescein) from DCFH ₂-DA using basic laboratory equipment. Next, the utility of DCFH ₂ was exemplified in a variety of cell-free and in vitro redox assay systems, including oxidant production by transition metals, photodynamic therapy, activated macrophages, and platelets, as well as the antioxidative capacity of different antioxidants. In cells, the concomitant use of DCFH ₂-DA and DCFH ₂ enabled the measurement and compartmentalized analysis of intra- and extracellularly produced oxidants, respectively, using a single read-out parameter. Furthermore, hepatocyte-targeted liposomes were developed to deliver the carboxylated derivative, 5(6)-carboxy-DCFH ₂, to hepatocytes in vivo. Liposome-delivered 5(6)-carboxy-DCFH ₂ enabled real-time visualization and measurement of hepatocellular oxidant production during liver ischemia-reperfusion. The liposomal 5(6)-carboxy-DCFH ₂ can be targeted to other tissues where oxidative stress is important, including cancer.