Indirect action to cell killing by SOBP carbon-ion beams

Materials and methods: Cell culture: The HSG cells were grown in E-MEM (SIGMA) supplemented with 10% FBS and antibiotics under a humidified air with 5% CO2 at 37 ̊C. The cells were seeded into T25 cm 2 flask (CORNING) at a concentration of 4 × 10 cells per flask for 48 h prior to irradiation. Irradiation and treatment with DMSO: Carbon ions (C) were accelerated by the HIMAC synchrotron to 290 MeV/nucleon. Depths in the SOBP beams were selected using a PMMA range shifter (116.10 and 143.84 mmH2O at middle and distal-end of SOBP, respectively). X-ray irradiations were performed using an X-ray generator (Shimadzu, Pantac HF-320S) operating at 200 kV and 20 mA, with a filter of 0.5 mm aluminum and 0.5 mm copper. The flasks were filled with 5 ml of medium containing different concentrations of DMSO ranging from 0 to 1.0 M for 1 h prior to irradiation. Colony formation assay: After irradiation, the cells were seeded into triplicate 60-mm plastic dishes at a density of ~100 living cells per dish and incubated for 14 days. The colonies were fixed with 10% formalin solution, stained with 1% methylene blue solution and colonies consisting of more than 50 cells were counted. The 10% survival level (D10 or LD90) was calculated from a dose– response curve fitted by an LQ equation. Calculation of the maximum protection by DMSO (DMSO method): The maximum degree of protection (DP), the concentration of DMSO that provides the maximum protection against cell killing, was calculated as well as our previous work [1–4]. Briefly, the maximum DP was calculated by an extrapolation of reciprocals of surviving fractions over those of DMSO concentrations. The DP was defined by the below equation, and regression curves were drawn in the plots of DP as a function of DMSO concentration.