Dichlorofluorescin diacetate

Fig. 1. Schematic representation of cell loading with 2, 7 -dichlorofluorescin diacetate and the oxidation events that occur when the phagocyte NADPH oxidase is activated.

Dichlorofluorescin diacetate (DCFH-DA Molecular Probes, Eugene, OR). 

Rota et al. [81,82] concluded that DCF fluorescence could not be a reliable assay of superoxide detection in cells because superoxide is formed during the DCFH oxidation by peroxidases. It has also been shown that DCFH is oxidized by heme, hemoglobin, myoglobin, and cytochrome c [83]. However, recent work by Caldefie-Chezet et al. [84] showed that the measurements of superoxide production by PMNs with the use of 2 -7 -dichlorofluorescin diacetate flow cytometry correlated with the data obtained by lucigenin- and luminol-amplified CL assays. 

Harvath and Terle 2, 7 -dichlorofluorescin diacetate. Cffi.i.lme.mbranQ,... 

The nonfluorescent probe 2, 7 -dichlorofluorescin diacetate is mixed with neutrophils in suspension to load the cells with the probe. Once 2, 7 -dichlorofluorescin diacetate is inside the cell, it is desacetylated by intracellular esterases forming the product 2, 7 -dichlorofluorescin, which is nonfluorescent and remains trapped within the cells (Fig. 1). When neutrophils are exposed to a stimulus that activates the oxidative burst, hydrogen peroxide (generated during neutrophil activation) reacts with the intracellular probe to form the fluorescent compound 2, 7 -dichloro-fluorescein. The fluorescent signal intensity, which is evaluated with a flow cytometer, is directly related to the oxidative metabolic activity of... 

LeBel CP, Ali SF, McKee M, et al. 1990. Organometal-induced increases in oxygen reactive species The potential of 2, 7 -dichlorofluorescin diacetate as an index of neurotoxic damage. Toxicol Appl Pharmacol 104 17-34. 

Two fluorescent probes, 2 ,7 -dichlorofluorescin diacetate and dihydrorhodamine 123, have been used for detecting intracellular reactive oxygen species production. Properties of an ideal agent for this purpose would include ... 

Yen et al. (2001) utilized the fluorescent dye 2 ,7 -dichlorofluorescin diacetate to measure the generation of reactive oxygen species in human umbilical vein endothelial cells. 17P-Oestradiol (54 /jM) pretreatment for 18 h or direct co-incubation significantly suppressed both ferf-butylhydroperoxide-and oxidised LDL-induced stimulation of the generation of reactive oxygen species. 

Recently Rota et al. (1999) demonstrated that dichlorofluorescein fluorescence cannot be used re-hably to measure 02 " in cells because 02- itself is formed during the conversion of dichlorofluorescin to dichlorofluorescein by peroxidases. The disproportionation of superoxide forms H2O2 which, in the presence of peroxidase activity, will oxidise more dichlorofluorescin to dichlorofluorescein with self-amplification of the fluorescence. Because the deacetylation of dichlorofluorescin diacetate, even by esterases, can produce H2O2, the use of this probe to measure H2O2 production in cells is problematic. 

Copper is stored by the liver in the storage protein metallothionein and excreted by the transport protein ceruloplasmin into the bile. When 10 rat hepatocytes were incubated with 50 piM Cu " for 2 h, the formation of reactive oxygen species as determined by oxidation of dichlorofluorescin diacetate to dichlorofluorescein increased from 90 5 intensity units in the controls to 412 9 intensity units in the metal-treated cells (Pourahmad and O Brien 2000). Malondialdehyde UV absorption increased from 0.048 0.006 to 0.662 0.012 units obtained at X ax = 532 nm (P <0.001). The ED50 concentrations found for Cu and Cd (i.e. 50% membrane lysis in 2 h) were 50 and 20 pM, respectively (Pourahmad and O Brien 2000). However, reactive oxygen species formation, GSH oxidation and hpid peroxidation were induced by Cu at these concentrations more rapidly than by Cd. The dechne of mitochondrial membrane potential though occurred at the same time and to the same extent for both metals. 

The esterified form of dichlorofluorescin, dichloro-fluorescin diacetate, crosses cell membranes and then undergoes deacet)dation by intracellular esterases. The resulting compound, dichlorofluorescin, is proposed to be trapped within the cell and susceptible to reactive oxygen species-mediated oxidation to the fluorescent compound, dichlorofluorescein. 

Intracellular peroxide levels were assessed using 2, T-dihydrodichlorofluorescein diacetate (H2DCFDA), an oxidation sensitive fluorescent probe. Once inside the cell, the probe is deacetylated by intracellular esterases forming 2, 7 -dichlorofluorescin (H2DCF), which is oxidized in the presence of a variety of peroxides, to a highly fluorescent compound, namely 2, 7 -dichlorofluorescein (DCF). 

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