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. DCFH2-DA (2′,7′-dichlorodihydrofluorescein diacetate) is the most widely used fluorogenic probe for the detection of general oxidative stress. However, the use of DCFH2-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 DCFH2 (2′,7′-dichlorodihydrofluorescein) from DCFH2-DA using basic laboratory equipment. Next, the utility of DCFH2 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 DCFH2-DA and DCFH2 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-DCFH2, to hepatocytes in vivo. Liposome-delivered 5(6)-carboxy-DCFH2 enabled real-time visualization and measurement of hepatocellular oxidant production during liver ischemia-reperfusion. The liposomal 5(6)-carboxy-DCFH2 can be targeted to other tissues where oxidative stress is important, including cancer.

Anticancer Photodynamic Therapy Properties of Sulfur‐Doped Graphene Quantum Dot and Methylene Blue Preparations in MCF‐7 Breast Cancer Cell Culture

“…In our experiment to measure ROS, the dye H 2 DCFDA was used, which is converted in the cytosol to 2′,7′‐dichlorodihydrofluorescein, a sensor that can detect a wide variety of ROS, including the superoxide anion radical (O 2 •‐ ), hydrogen peroxide, the hydroxyl radical and SO . As this dye detects a broad spectrum of ROS, it is possible that the ROS we measured stem from both Type I and Type II photosensitized oxidation reactions.…”

“…), hydrogen peroxide, the hydroxyl radical and SO (55)(56)(57). As this dye detects a broad spectrum of ROS, it is possible that the ROS we measured stem from both Type I and Type II photosensitized oxidation reactions.…”

An Endogenous Reactive Oxygen Species (ROS)‐Activated Histone Deacetylase Inhibitor Prodrug for Cancer Chemotherapy

“…The non‐fluorescent DCF‐DA reagent is converted into the fluorescent DCF in the presence of intracellular ROS. Indeed, in the DCF‐DA assay we observed significantly increased fluorescence signal only in HeLa cells and not in HEK‐293T cells (Figure S6) . The HeLa cells showed sixfold higher fluorescent intensity than HEK‐293T cells.…”

“…Indeed, in the DCF-DAa ssay we observed significantly increasedf luorescence signal only in HeLa cells and not in HEK-293T cells ( Figure S6). [42,43] The HeLac ells showed sixfoldh igher fluorescent intensity than HEK-293T cells. In HEK-293T cells, the fluorescencei ntensity of DCF-DA was similart othat in PBS.…”

Cytotoxic effect of <em>Drimia maritima</em> bulb extract and induction of mitochondrial apoptotic signaling in human breast cancer cells, MCF-7 and MDA-MB-468

“…The MarkerGene™ Live Cell Fluorescent ROS Detection Kit (Marker Gene Technologies) was used to measure ROS generation in MCF-7 and MDA-MB-468 exposed to D. maritima . 14 This kit uses a cell-permeable substrate (DCFH-DA) as a fluorogenic marker for ROS detection. Briefly, after incubation with DCFH-DA, cells were harvested and washed with HBSS and analyzed by a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA).…”