Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader

Oxidative stress (OS) has been implicated in various degenerative diseases in aging. In an attempt to quantify OS in a cell model, we examined OS indu ced by incubating for 30 min with various free radical generators in PC12 c ells by using the dichlorofluorescein (DCF) assay, modified for use by a fl uorescent microplate reader. The nonfluorescent fluorescin derivatives (dic hlorofluorescin, DCFH), after being oxidized by various oxidants, will beco me DCF and emit fluorescence. By quantifying the fluorescence, we were able to quantify the OS. Our results indicated that the fluorescence varied lin early with increasing concentrations (between 0.1 and 1 mM) of H2O2 and 2,2 '-azobios(2-amidinopropane) dihydrochloride (AAPH; a peroxyl radical genera tor). By contrast, the fluorescence varied as a nonlinear response to incre asing concentrations of 3-morpholinosydnonimine hydrochloride (SIN-1; a per oxynitrite generator), sodium nitroprusside (SNP; a nitric oxide generator) , and dopamine. Dopamine had a biphasic effect; it decreased the DCF fluore scence, thus acting as an antioxidant, at concentrations <500 mu M in cells , but acted as a pro-oxidant by increasing the fluorescence at 1 mM. While SNP was not a strong pro-oxidant, SIN-1 was the most potent pro-oxidant amo ng those tested, inducing a 70 times increase of fluorescence at a concentr ation of 100 mu M compared with control. Collectively, due to its indiscrim inate nature to various free radicals, DCF can be very useful in quantifyin g overall OS in cells, especially when used in conjunction with a fluoresce nt microplate reader. This method is reliable and efficient for evaluating the potency of pro-oxidants and can be used to evaluate the efficacy of ant ioxidants against OS in cells. (C) 1999 Elsevier Science Inc.