Medium was changed every 2 days. Seven days old cells were exposed to water samples, negative and positive controls (500 μM hydrogen peroxide) for 20 min and afterwards cell suspensions were prepared with 0.25% trypsin-EDTA solution, passed through injection needle for several times to achieve single cell suspension and finally resuspended in DMEM medium, supplemented with 10% FCS medium. The dye-exclusion test with Trypan blue was used to examine the viability of cells before the comet assay was performed.27
multilayer culture