Triad multi mode microplate reader

The apoptosis assay was performed using Cell Death Detection ELISA^PLUS (Roche), which is an ELISA assay for the quantitative determination of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes). The cells were reverse transfected with RNA oligonucleotides in 96-well plates and treated with cisplatin for 24 h at 48 h after the reverse transfection, exactly as described above (section: cell culture, reverse transfection, and apoptosis induction). Following the cisplatin treatment (72 h after reverse transfection), the procedure was carried out according to the manufacturer’s protocol and the absorbance of each well was measured with ELISA plate reader (Dynex Technologies Triad Multi-Mode Microplate Reader) at 405 nm. For each group, the average background value was subtracted from the average absorbance measurement, followed by the calculation of the enrichment factor. The factor represented the ratio of each cells group to untransfected and cisplatin-untreated cells’ control group. Samples were prepared in triplicate and absorbance measurement for each sample was done in duplicate.

To performed cell viability assay, the cells were reverse transfected with RNA oligonucleotides in 96-well plates and treated with cisplatin for 24 h at 48 h after the reverse transfection, exactly as described above (section: cell culture, reverse transfection, and apoptosis induction). Following the cisplatin treatment (72 h after reverse transfection), the medium in each well was aspirated and replaced with 100 µl of the mixture containing: RPMI-1640 medium without phenol red (Gibco^®); XTT solution (BioShop Canada Inc; dissolved in PBS from Gibco^®) at the final concentration of 200 µg/ml and PMS (phenazine methosulfate) solution (Sigma-Aldrich; dissolved in PBS from Gibco^®) at the final concentration of 2 µg/ml. After the incubation at 37 °C for 3 h in the dark, the absorbance of each well was measured with ELISA plate reader (Dynex Technologies Triad Multi-Mode Microplate Reader) at 450 nm. All cell groups were compared against untransfected and cisplatin-untreated cells’ control group (considered as 100% viable). Samples were prepared in triplicate.

Cell viability assay was performed using: XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt; BioShop Canada Inc.) dissolved in phosphate-buffered saline (PBS) solution (Gibco^®); phenazine methosulfate (PMS) solution (Promega); and RPMI-1640 medium without phenol red (Gibco^®).
For the XTT assay, cells were seeded at 6 × 10³ cells/100 µl medium/0.32 cm² growth area in 96-well plates, grown overnight, and treated with morin or cisplatin for 24 h and/or 48 h. Concentrations of drugs’ solvents were corrected in all wells (including control wells) to the constant level, corresponding to the highest used concentration of a particular solvent. Following the treatments, the medium in each well was replaced with 100 µl of the mixture of RPMI-1640 medium without phenol red, XTT solution (at the final concentration of 200 µg/ml) and PMS solution (at the final concentration of 2 µg/ml), prior to incubation at 37 °C for 3 h in the dark. The absorbance of each well was measured at 450 nm with ELISA plate reader (Dynex Technologies Triad Multi-Mode Microplate Reader). All treated cells were compared against control cells (considered as 100% viable). IC₅₀ (half maximal inhibitory concentration) values were determined for each drug at each treatment time. Samples were prepared in triplicate.