5 fluoruracil

Cell viability of the ES-D3 cells was executed as before [28 (link)]. 500 cells per well were plated in a 96-wells plate (Greiner Bio-One) and were kept warm at 37 ⁰C and 5% CO₂ for two hours. Cells were exposed in six replicates to 200 μl of LIF-containing medium including the OPs in concentrations ranging from 330 μM to 0,33 μM or 0 μM or including the negative control penicillin G (500 μg/mL; Sigma-Aldrich), the positive control 5-fluoruracil (0.1 μg/mL; Sigma-Aldrich), or the vehicle control DMSO (0.25%; Sigma-Aldrich). The exposure medium was refreshed at identical final concentrations after three days of exposure under 37 ⁰C and 5% CO₂. Following incubation for a further two days, the exposed plates were prepared for the viability fluorescence measurements by replacing 100 μl of solution by 20 μl of CellTiter-Blue reagent (Promega, Leiden, The Netherlands) per well [29 ]. After 2 h of incubation the extinction values were determined at 544_Ex/590_Em nm on the SpectraMax® M2 spectrofluorometer (Molecular Devices, Berkshire, United Kingdom). Viability levels were calculated relative to the DMSO control (in %). For each test compound the average and standard deviation of the six replicates of each experiment (n = 3) were analysed using PROAST v67.0 as described in section 2.9. This was used to determine the concentration for which 50% of the cells were viable (IC₅₀ values).