Isolation and culture of mouse primary BMMs

Granulocyte-macrophage colony stimulating factor (GM-CSF) is required to induce hematopoietic cell differentiation into macrophages. L929 cells can produce GM-CSF. L929 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 5% CO₂ at 37 °C for 7 days (replacing culture medium at day 4 with fresh complete DMEM). L929 cells were then harvested and implanted with 4.7 × 10⁵/bottle and cultured in the presence of DMEM-Gluta MAX medium. After 7 days, the medium was sterilized by filtrating through a 0.22 μm filter and stored at −20 °C, which used as L929-conditioned medium^40–42.

C57BL/6J mice aged 4 weeks were sacrificed by cervical dislocation at the time of bone marrow (BM) harvest. BM cells were extracted from the femurs by flushing with culture medium. BM cells were cultured in the presence of culture medium supplemented with 10% FBS and 10% L929-conditioned medium (replacing culture medium at day 3 and 5)^40–42. After 5 days, mice primary BMMs were exposed to different doses of IR.