Spectrofluorometry assays for oxidative stress and apoptosis, with cell viability on the same microplates: A multiparametric analysis and quality control

Our objective was to compare neutral red (NR) tests performed separately and on the same microplates as hydroethidine, H2DCF-DA and YO-PRO®-1 assays, and to compare toxicity ratios calculated with the two methods. Human trabecular meshwork cells (HTM-3) were exposed to different concentrations of benzalkonium chloride (BAC), to PBS and to diluted solutions of latanoprost and bimatoprost. Microplate fluorescence spectrophotometry assays were performed: NR uptake for cell viability evaluation, hydroethidine and H2DCF-DA for reactive oxygen species (ROS), and YO-PRO®-1 for apoptosis. The four NR assays presented identical toxicological profiles and correlation coefficients between them were close to one. There was no difference between toxicity ratios for ROS assays calculated by the two methods, with high correlation coefficients. However, in the apoptosis assay, ratios calculated with the double staining technique were more accurate. Thus, it is possible and recommended to perform NR assays on the same microplates as the other assays. This double staining procedure could constitute a quality control procedure and would allow multiparametric analysis.

► We compared neutral red assay performed separately and after other assays.
► Test solutions presented same toxicity with the different neutral red assays.
► Toxicity ratios over neutral red on same and different microplates were identical.
► Double staining would allow quality control and multiparametric analysis.