2.4. Sister Chromatid Exchange Assay

CHO synchronized in G1 phase cells were subcultured into four P-60 petri dishes at a density of approximately 10⁶ cells per dish in medium containing 5-bromo-2′-deoxyuridine (BrdU) (Sigma), with a final concentration of 10 μM for two cycles. Then, 0.2 μg/mL colcemid (Gibco, Invitrogen, Grand Island, NY, USA) was added to replicate petri dishes for four-hour intervals beginning at 26 or 32 h for SCE, depending upon the peak number of second passage mitosis SCE were scored in cultures containing the peak number of second-passage mitosis. Cells were harvested during metaphase, trypsinized, and then suspended in 2 mL of 75 mM KCl solution warmed to 37 °C and placed in a 37 °C water bath for 20 min. A fixative solution of 3:1 methanol to acetic acid was added to the samples according to the standard protocol [34]. Fixed cells were dropped onto slides and allowed to dry at room temperature. Differential staining of metaphase chromosomes was completed using the fluorescence plus Giemsa (FPG) technique with Hoechst 33,258 dye, followed by UVR exposure [35]. Differentially stained metaphase chromosome images were scored under a Zeiss Axioskop microscope equipped with a SPOT CCD camera RT 2.3.1 (Diagnostic Instrument, Inc., Sterling Heights, MI, USA) and SPOT basic software. A minimum of 50 metaphase cells were scored for each experiment. Data presented are the mean of SCE frequency per chromosome.