Measurement of reactive oxygen species in cultured primary rat hepatocytes

Basically, the standard operating procedure outlined in this document consists of the following steps, namely, (i) preparation of the cells for the DCFH-DA assay, (ii) DCFH-DA test procedure, (iii) processing of the results. Practical details are provided for each of these steps and are followed by some useful tips based upon our own hands-on experience. The fluorescent assay DCFH-DA is based on the ability of the non-fluorescent lypofilic DCFH-DA probe to penetrate viable cells, where it is being deactylated by the presence of intracellular esterases and trapped within the cells. Upon exposure of the cells to a stimulus that generates the oxygen metabolic burst and subsequently the production of ROS, the probe is oxidized and starts to emit an intense green fluorescence. This assay allows the determination of the oxidative stress induction potential of chemical substances. As such, after incubation with the probe, the cells are exposed to the selected substances and the respective emitted fluorescence can be measured for a period of 30 min. The increase in fluorescence is proportional to the amount of ROS that are being formed within the cells. The results can be read by the use of a multiwell scanning fluorimeter (plate reader) using an 485-495nm emission filter and an 520-530nm excitation filter. ROS production is expressed as a ratio of treatment versus control. This method provides a sensitive, reproducible and integrated signal of both attached cells and cells in suspension.