Cells were irradiated with TITAN x-ray irradiator with 200 kVp, 20 mA, 0.5 cm of Al and Cu filter (Shimadzu, Japan). Heavy ion treatment was performed by HIMAC (Heavy Ion Medical Accelerator in Chiba). The accelerated ions used in this study were carbon ions (290 MeV/n), neon (400 MeV/n), silicon (490 MeV/n), argon (500 MeV/n), and iron ions (500 MeV/n). The details concerning the beam characteristics of carbon-ion beams, biological irradiation procedures, and dosimetry have been described elsewhere [19 (link),20 (link)]. We used several kinds of beams having different LET values, using Lucite absorbers with various thicknesses to change the energy of the beams. At the sample position, we estimated the LET values of carbon (13, 30, 50, 70 keV/μm), neon (31, 70, 120 keV/μm), silicon (55, 150, 250 keV/μm), argon (100 keV/μm), and iron (200 keV/μm). Taking fragmentations into consideration, dose was calculated from fluence [21 (link)-23 ]. Asynchronously dividing cells cultured in T12.5 flasks were irradiated at room temperature. For chemical treatment, cycling cells in T12.5 culture flasks were exposed to series of concentration of bleocin, a single component of bleomycin family group A (Calbiochem, Japan), which induces DNA strand breaks, camptothecin (CPT, Sigma, Japan) which is a Topoisomerase I inhibitor, mitomycin C (MMC, Funakoshi, Japan) which induces DNA crosslink, or cisplatin (Nippon Kayaku, Japan) which induces DNA crosslink for 1 hour at 37°C.
After exposure to ionizing radiation or chemical treatment, cells were trypsinized and re-plated in P-100 cell culture dishes. HeLa and U87-MG cells were cultured for 10 to 14 days, and U-CH1-N cells were kept in an incubator for 3 to 4 weeks. Plating efficiency of U-CH1-N, U87-MG, and HeLa cells were 4.8%, 32%, and 70%, respectively. After colonies were formed, cells were fixed with 100% ethanol and stained with crystal violet solution. Colonies were observed under microscope and colonies containing more than 50 cells were counted as survivors. Cell survival assay was carried out 2 to 4 times independently. Radiation exposed cell survival curves were fitted with linear quadratic model by PRISM5 software on MacOSX10.6. Error bars indicate standard error of the means.
After exposure to ionizing radiation or chemical treatment, cells were trypsinized and re-plated in P-100 cell culture dishes. HeLa and U87-MG cells were cultured for 10 to 14 days, and U-CH1-N cells were kept in an incubator for 3 to 4 weeks. Plating efficiency of U-CH1-N, U87-MG, and HeLa cells were 4.8%, 32%, and 70%, respectively. After colonies were formed, cells were fixed with 100% ethanol and stained with crystal violet solution. Colonies were observed under microscope and colonies containing more than 50 cells were counted as survivors. Cell survival assay was carried out 2 to 4 times independently. Radiation exposed cell survival curves were fitted with linear quadratic model by PRISM5 software on MacOSX10.6. Error bars indicate standard error of the means.
Free full text: Click here
