Reactive oxygen species are formed in cell culture media.

When studying the effects of oxidative stress on mammalian and yeast cells, we observed that the media without cells generated reactive oxygen species. Mammalian cell media such as RPMI 1640 medium with Glutamax-I (GibcoBRL, Cat. No. 31996) and Dulbecco’s Modified Eagle Medium with Glutamax-I, sodium pyruvate and pyridoxine (GibcoBRL, Cat. No. 31966; DMEM), as well as the yeast extract-peptone-glucose medium containing 1% yeast extract, 1% peptone and 2% glucose were found to oxidize 5 M dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydrorhodamine 123 (H2R123), and to generate an OH-type adduct of 5,5 -dimethyl-1-pyrroline N-oxide (DMPO) under aerobic conditions. Spin trapping demonstrated formation of an OH-type adduct. Electron spin resonance spectra originated from decomposition of O2 –. adducts of DMPO rather than from trapping of OH, since their intensity was significantly reduced in the presence of 50 g/ml superoxide dismutase. The rate of oxidation of H2DCFDA and DHR123 as well as of DMPO adduct formation was higher for DMEM medium than for RPMI 1640 medium and was significantly attenuated in complete media containing 10% fetal calf serum (FCS). Horseradish peroxidase (HRP) considerably potentiated the rate of H2DCFDA and H2R123 oxidation, indicating that hydrogen peroxide was the main agent responsible for the oxidation of these fluorogens. Vol. 47 No. 4/2000