Cisplatin

Twenty-four hours before adding the tested compounds, all cell lines were seeded in 96-well plates (Sarstedt, Germany) in appropriate media with 0.5 × 10⁴ cells per well for A549 cell line, 0.75 × 10⁴ cells per well for MCF-7 cell line and 1.0 × 10⁴ cells per well for LoVo, LoVo/DX and BALB/3T3 cell lines. All cell lines were exposed to each tested agent at different concentrations in the range 100–0.001 μg/mL for 72 h. The cells were also exposed to the reference drug cisplatin (Teva Pharmaceuticals, Poland) and doxorubicin (Accord Healthcare Limited, UK). Additionally, all cell lines were exposed to DMSO (solvent used for tested compounds) (POCh, Poland) at concentrations corresponding to those present in dilutions of tested agents. After 72 h sulforhodamine B assay (SRB) was performed [64 (link)].

Twenty four hours before adding the tested compounds, each of the cell lines was seeded in 96-well plastic plates (Sarstedt, Numbrecht, Germany) in an appropriate medium at a density (10⁴ cells/well), except A549 cell line (0.25 × 10⁴/well), and MCF7 cell line: (0.75 × 10⁴/well). The selected cell lines were exposed to each of the tested chemical compounds at four different concentrations in the range of 100 to 0.1 µg/mL for 72 h. As a reference, cisplatin (Teva Pharmaceuticals, Poland) was used, and DMSO (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) served as a solvent control at concentrations corresponding to these present in the dilutions of the tested compounds. For adherent cells, a sulforhodamine B assay (SRB), and for leukemic—an MTT assay was performed.

10,000 of cells were seeded to each well of 96-well plate for overnight. On the next day, doxorubicin (Sigma) or cisplatin (Teva) were added at different concentration for 48 h; 6 wells per sample were used. Then 10 μl of 5 mg/ml Triazolyl Blue solution was added to each well for 4 h at 37 °С. After removing MTT containing medium, 150 μl isopropyl alcohol (supplemented with 40 mM HCl and 0,1% NP-40) was added to dissolve MTT-formazan salt. The absorbance at 570 nm and 630 nm was measured using BioRad iMark microplate reader (BioRad, USA).