Celltiter96 non radioactive proliferation assay

Cell cultures were kept under standard conditions (5 % CO₂, 37 °C, 90% humidity) in an RPMI medium with 10% FCS, 100 µg/mL streptomycin, and 100 U/mL penicillin (Life Technologies, Darmstadt, Germany). Reference cisplatin (Sigma, Taufkirchen, Germany) was dissolved freshly in 0.9% NaCl solution at a concentration of 1 mg/mL, and diluted appropriately. Described metal(II) complexes and their ligands were dissolved in DMSO. Platinum-resistant A2780 and SKOV3 cells were established as described [18 (link)]. IC₅₀ values were determined using the CellTiter96 non-radioactive proliferation assay (MTT assay, Promega, Walldorf, Germany). A total of 5000 cells were allowed to attach per well of 96-well plates for 24 h and treated for 48 h with different concentrations of the substances (ligands tests: 0, 1, 10, 50, 100, 500, 1000 µm) and for cisplatin and metal complexes from 0 to 100 µM (0.1, 1, 5, 10, 50, 100 µM). Each measurement was performed in triplicate and repeated three times. The amount of metabolic active cells was quantified using the MTT assay. Relative values compared to the mean of medium controls were calculated after background subtraction. Non-linear regression analyses were conducted in GraphPad 5.0 software using the Hill slope.

Following uc.8+ silencing with siRNAs-2 and -3, BlCa cell proliferation was measured using the CellTiter 96 nonradioactive proliferation assay (Promega). J82 cells were seeded (500 cells per well) in 96-well plates and assayed 0, 12, and 24 h later according to the manufacturer's protocol.

Cells (ASCs or fibroblasts: 3 × 10⁵) were embedded in the fibrin gel (100 μL) and cultivated in culture medium for 24 hours before medium was discarded and proliferation of cells was measured using a CellTiter96 Non-Radioactive Proliferation Assay (Promega Corporation, Madison, WI). Fibrin clots with no cells were used as negative control. Cell number was evaluated according to manufacturer's protocol: supernatants were discarded and 45 μL dye solution was added to 300 μL culture medium. After 1.5 hours of incubation at 37°C in a humidified atmosphere with 5% CO₂ stop solution was added. After one hour of incubation 100 μL of the supernatants was transferred in triplicates on a 96-well plate and absorbance was measured at 555 nm on a Wallac 1420 VICTOR2 plate reader (PerkinElmer, Waltham, MA, USA). Further proliferation assays were performed on day 7 and day 14 of the experiment. Additionally, fibrin clots with no cells were used for negative control measurement. Fibrin clots with cells from at least 4 different donors were analyzed in independent experiments.