H. Wang et Ja. Joseph, Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader, FREE RAD B, 27(5-6), 1999, pp. 612-616
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.