Oxidation of fluorogenic probes detecting reactive oxygen species in the extracellular medium

Anthocyanins are natural antioxidants that, at low concentrations, are not cytotoxic and do not inhibit cell proliferation in vitro and change color in response to pH changes. An attempt to use anthocyanins as an extracellular indicator instead of Phenol Red showed that changes in the absorption spectrum and an oxidation-sensitive fluorescence band are altered with time in the culture of SKOV-3 cells, but, surprisingly, the changes are more pronounced in the absence than in the presence of cells. To examine the generality of this phenomenon, oxidation of fluorogenic probes used for the detection of reactive oxygen species (ROS), dihydrorhodamine (H₂R 123), dihydroethidium (DHE), and 2',7'-dichlorodihydrofluorescein diacetate (H₂DCF-DA) in cell culture and cell-free media was compared. Comparable or higher oxidation rates in cell-free medium were found for all probes. These results point to autoxidation of redox-active components of the medium as the main cause of changes in the properties of cell culture media and suggest that the intracellular oxidation of the probes may also be partly due to their autoxidation.