Lucia comet 4

The comet assay analysis was performed with an Eclipse fluorescence microscope (Nikon, Tokyo, Japan) attached to a COHU 4910 video camera (Cohu, Poway, CA, USA), using the computer-based image analysis system, Lucia Comet 4.51 (Laboratory Imaging, Praha, Czech Republic). The microscope is equipped with a UV filter block consisting of an excitation filter (λ = 359 nm) and a barrier filter (λ = 461 nm). DNA repair efficiency was measured after 60 min incubation with 10 µM DDRI-18 or 120 min of incubation with 10 µM A12B4C3 and another 2 h exposure to CDDP and VP-16 alone and in combination. The DNA damage repair process was evaluated after 120 min of incubation in a drug-free medium. After that, cells were collected at time points from 0 to 2 h. As a time 0 (the initial level of DNA damage) we used cells collected directly after drug treatment. The level of DNA damage from the remaining time points was compared with that obtained for time 0. The level of DNA damage was measured as the percent of DNA in the comet tail.