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5 cells/well in selection medium and incubated at 37°C overnight. The following day, cells were washed twice to remove serum and then incubated in 300 μL/well DMEM with insulin, transferrin and selenium supplements (Sigma, UK) added. Test compounds ((R,R)-2b, (R)-1) at 0, 1, 5 and 10 μM were added to duplicate wells. After a further 24 h, media were removed for zymographic analysis and the cells were washed in PBS. The cells from each well were harvested by trypsinization, resuspended in PBS and mixed 1:1 with trypan blue (0.4% w/v, Sigma, UK). After 5 min, a sample of cell suspension from each well was removed and introduced into a haemocytometer. The total number of cells in the upper grid was counted and a separate count was taken of the cells, which had taken up the blue dye, that is, the dead cells. The process was repeated by counting the cells in the lower grid. Percentage toxicity for each compound at the different concentrations was calculated as a mean of the values for duplicate wells, after subtracting the values for the wells without either compound
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