96 well microtiter tissue culture plates

C6 Rat glioma and NIH/3T3 mouse embryonic fibroblast cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma, Deisenhofen, Germany) supplemented with 10% fetal calf serum (Gibco, Paisley, UK). A549 Human lung adenocarcinoma cells were incubated in 90% RPMI supplemented with 10% fetal bovine serum (Gibco). All media were supplemented with 100 IU/mL penicillin-streptomycin (Gibco) and cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. Exponentially growing cells were plated at 2 × 10⁴ cells/mL into 96-well microtiter tissue culture plates (Nunc, Roskilde, Denmark) and incubated for 24 h before the addition of the drugs (the optimum cell number for cytotoxicity assays was determined in preliminary experiments). The stock solutions of the compounds were prepared in dimethyl sulfoxide (DMSO; Sigma Aldrich, Poole, UK) and further dilutions were made with fresh culture medium (the concentration of DMSO in the final culture medium was <0.1% which had no effect on the cell viability).

This is a colorimetric assay that measures the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,4,diphenyltetrazolium bromide (MTT) by mitochondria [46 (link)]. All final compounds were tested against human breast adenocarcinoma cell line (MCF-7), human lung carcinoma cell line (A549) and mouse embryooblast cell line (NIH/3T3) to determine their cytotoxic profile. Cancer cells were cultured in RPMI medium (Hyclone, Thermo Scientific, Waltham, MA, USA) at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. The studied cells were situated at 2 × 10⁴ cells into each well of 96-well microtiter tissue culture plates (Nunc, Denmark) and incubated for 24 h. After that, test compounds were dissolved in DMSO and added to culture wells at varying concentrations (85–1000 µM). After a 24 h incubating period, 20 mL MTT solution (5 mg/mL MTT powder in PBS) was added to each well and the cells were incubated for 4 h at 37 °C. The purple formazan crystals produced were dissolved in DMSO and the absorbance was read by ELISA reader (OD570 nm). The percent values of cell proliferations were calculated relative to controls, whose cell proliferations were accepted as 100% [47 (link)].

C6 Rat glioma and NIH/3T3 mouse embryonic fibroblast cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma, Deisenhofen, Germany) supplemented with 10% fetal calf serum (Gibco, Paisley, UK). A549 Human lung adenocarcinoma cells were incubated in 90% RPMI supplemented with 10% fetal bovine serum (Gibco, Paisley, UK). All media were supplemented with 100 IU/mL penicillin-streptomycin (Gibco, Paisley, UK) and the cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. Exponentially growing cells were plated at 2 × 10⁴ cells/mL into 96-well microtiter tissue culture plates (Nunc, Roskilde, Denmark) and incubated for 24 h before the addition of the drugs (the optimum cell number for cytotoxicity assays was determined in preliminary experiments). The stock solutions of the compounds were prepared in dimethyl sulfoxide (DMSO; Sigma Aldrich, Poole, UK) and further dilutions were made with fresh culture medium (the concentration of DMSO in the final culture medium was <0.1% which had no effect on the cell viability).