Briefly, after permeabilization with 0.1% saponin (15 minutes), the cells were stained with Click-iT reaction mix for 30 minutes (dark room). test was applied for each time point (red for low LET and blue for high LET; *< .05, **< .01, and ***< .001). Western Blotting Analysis Following irradiation, cells were detached from the flasks at different time points, centrifuged at 845for 5 minutes, and the cell pellets were mixed with T-PER lysis buffer supplemented with a protease and phosphatase inhibitors cocktail (ref KT203 78440; Thermo Fisher). IL20RB antibody This cell lysis step was followed by addition of Laemmli buffer and KT203 a denaturation at 100C. The extracted sample was then separated by SDS-PAGE and transferred to a nitrocellulose membrane according to Hamdi et al.22 Membranes were analyzed against anti-H2AX phospho-serine 139 (clone JBW301; Merck, Fontenay-sous-Bois, France), anti-GAPDH (MA5-15738; Fisher, Illkirch, France), and anti-p21 (2947; Cell Signaling, Denver, Colorado). Membranes were then incubated with HRP-conjugated secondary antibody (mouse or rabbit; 1:10000; GE Healthcare). The membranes were treated with electrochemiluminescence reagent (Merck KGaA, Darmstadt, Germany) before exposure to hyperfilms (VWR, Fontenay-sous-Bois, KT203 France). The films were developed and scanned as JPEGs using a GS 700 Bio-Rad scanner (Bio-Rad, Hercules, CA). Micronucleus Test The cells were plated on 10-mm-diameter glass coverslips placed in 24-well plates so that they reach subconfluence at the time of analysis. About 22 hours before harvest and 4 hours after irradiation, cytochalasin B (Sigma-Aldrich) was added at a concentration of 3 g/mL in culture medium. For the analysis of micronuclei, the cells were washed with PBS and fixed in cold acid acetic (10% vol/vol) in methanol solution for 20 minutes. The coverslips were mounted on glass slides with Prolong Gold Anti-Fade reagent with DAPI KT203 (Invitrogen, Paris, France) which allowed KT203 staining of the DNA. For each experimental point, 500 binucleated cells were analyzed per slide, for at least 3 slides. The micronuclei were scored only in binucleated cells where the 2 nuclei had similar size and staining intensity and did not present nuclear condensation or any other morphology abnormalities. The micronuclei were considered when they were about 1/3 to 1/16 of the size of nucleus and presented similar staining intensity. The experiments were repeated at least 3 times and data expressed as mean SEM. A one-way ANOVA test was applied to assess significance at the .05 level. Results Clonogenic Survival Is Reduced With C-Ions as Compared to X-Ray Radiation The clonogenic survival was calculated for the 4 chondrosarcoma cell lines with increasing doses of X-rays or C-ions. Eighteen hours following irradiations at culture confluency, the cells were seeded in culture flasks at low density and the plating efficiencies of SW1353, CH2879, OUMS27, and L835 cells were 0.17 0.02, 0.51 0.08, 0.32 0.05, and 0.34 0.04, respectively. The cells were kept in a humidified incubator at 5% CO2 and 2% O2 for at least 8 days, until large clones could be observed but without cells merging from different clones. Clones with more than 50 cells were counted and survival curves were fitted by Linear-Quadratic (LQ) equation in case of X-rays and linear model in case of C-ions irradiations (Figure 1). Open in a separate window Figure 1. Comparison of clonogenic survival of 4 chondrosarcoma cell lines irradiated with different radiation qualities. The surviving fractions of chondrosarcoma cells irradiated with 225 kV X-rays (blue squares), 28 keV/m C-ions (red squares), and 73 keV/m C-ions (green squares). Four chondrosarcoma cell lines were plotted with the same irradiation conditions: (A) (SW1353), (B) (CH2879), (C) (OUMS27), and (D) (L835). The symbols and the bars corresponded respectively to the means and standard errors from at least 3 independent experiments. The data were fitted with the linear quadratic equation in case of X-rays irradiations, and with a linear equation for C-ions irradiations, as explained in the corresponding paragraph of the Materials and Methods. The plots were obtained from the CS-cal software, which allowed the calculation of survival and biological effectiveness parameters (Table 1). Considering X-rays, the L835 cell line was observed as the most sensitive with a D10 of 4.16 0.11 Gy; and the OUMS27 and SW1353 cell lines were the most resistant with a D10 of about 6.7.