Cell culture dish

A mammalian cell line, Chinese Hamster Ovary (CHO)-K1 was obtained from RIKEN Bio Resource Center, Japan (RBC0285). This type of cell line was selected because it does not exhibit HRS^{21 (link)}. The cells were maintained in Dulbecco’s modified Eagle’s (DMEM, Sigma Life Science) supplemented with 10% fetal bovine serum (FBS, Equitech-Bio Inc.) and 1% penicillin/streptomycin (Sigma Life Science) at 37 °C in humidified 95% air and 5% CO₂.
To investigate cell responses after a long-term exposure, five days prior to irradiation 4 × 10⁵ cells were seeded onto the cell culture dish with 60 mm diameter (Nippon Genetics) to obtain the cells under plateau phase. In parallel, to quantify the dependence of SLDR rates on cell-cycle distribution, we prepared two cell-cycle distributions for plateau phase and logarithmic growth phase five days and one day after seeding, respectively.

After exposure to the regimen equivalent to 3.0 and 6.0 Gy/h, irradiated cells were trypsinized immediately and the appropriate number of cells was reseeded into a cell culture dish with 60 mm diameter (Nippon Genetics). Culture medium was exchanged every two days and the cells were cultured for 10–14 days. The colonies were fixed with methanol and were stained with 2% Giemsa solution (Kanto Chemical Co. Inc.) Survival fraction was obtained from the ratio of colony number of irradiated cells to that of non-irradiated cells (control cells). The plating efficiency for control cells was 38.8 ± 9.2% (mean ± standard deviation), which was given by 27 dishes (the assay was performed three times for each dose-rate, and three dishes were used in one assay).