Reagenta

The effect that the compounds 1a–6a exert on the melanoma cell number (in cell culture) was tested on a MEW155 human melanoma tumor cell line obtained from the Maria Skłodowska-Curie Memorial Cancer Centre and Institute of Oncology in Warsaw. The cells were plated in 24-well plates (5000 cells per well) and incubated for 24 h in Eagle’s medium containing 10% fetal bovine serum and 1% solution of penicillin and streptomycin. After the cells had reached 80% confluence, the tested compounds (dissolved in 1% DMSO) were added at a concentration of 50 μM. Control cells were treated with 1% DMSO only. After 96 h of incubation, the cell suspension was first lysed in lysis buffer (ReagentA, ChemoMetec A/S) and then stabilized in stabilization buffer (ReagentB, ChemoMetec A/S). The number of dead cells was quantified in a propidium iodide cassette with the use of a NucleoCounter^® NC-100™ (ChemoMetec A/S) cell counter. The tests were performed in triplicate. The results were normalized so that the values obtained for the control were 100%.

PBMCs were quantified with an automated device based on propidium iodide (PI) staining and exclusion, the NucleoCounter NC-100 (ChemoMetec, Denmark). For total cell count, PBMCs were lysed and stabilized with reagent A and reagent B (ChemoMetec, Denmark), respectively, to expose all nuclei to PI staining. Counting of dead cells was performed without lysis treatment, and viability was calculated as the proportion between living cells and the total cell count and was expressed as a percentage.