137cs irradiator

Total BM was collected from donor mice by flushing femurs and tibiae with sterile PBS supplemented with 10% FCS (Invitrogen) and penicillin (50 U/ml)/streptomycin (50 µg/ml; Invitrogen). Recipient mice were lethally irradiated with two doses of 4.5 Gy, divided by 3 h minimally, using a ¹³⁷Cs irradiator (CIS Bio International). After the last irradiation dose, mice received an intravenous injection of total volume of 300 µl per mouse containing 5 × 10⁶ donor BM cells and 2 × 10⁵ spleen cells of the recipient strain to induce acute radioprotection. Three groups were created: BM from NLRX1 KO mice into WT recipients (KO → WT), BM from WT mice into NLRX1 KO recipients (WT → KO), and BM from WT mice into WT recipients (WT → WT).
For 6 wk after transplantation, mice received sterile acidified tap water (12 mM HCl) that contained 0.16% neomycin sulfate (Sigma-Aldrich). Functional BM engraftment was determined by expression analysis of CD45.1 (WT C57BL6/J animals) and CD45.2 (NLRX1 KO animals) by peripheral leukocytes using a FACSCalibur (BD Biosciences) flow cytometer. Chimeras with donor BM engraftment of >75% or higher were used for further experiments. Note that this approach was not used for WT → WT control BM transplantation animals. No gross hematological abnormalities were detected in all animals after transplantation.

RKO human colorectal cancer cells and A549 human lung cancer cells were used. The cells were grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were cultured in 25 cm² plastic tissue culture flasks at 37℃ in a humidified 5% CO₂/95% air atmosphere. When the cells were in exponential growth phase at a cell density of 3×10⁶ cells/ 25 cm² flasks, cells were irradiated with 10 Gy of γ-rays at a dose rate of 5.0 Gy/min with a 137 Cs irradiator (Cis biointernational IBL437C, France).