2.3. Differentiation of murine bone marrow‐derived macrophages and co‐culture experiments with GL261 cells

Bone‐marrow cells were obtained by flushing the tibia and femurs of WT and Acod1 KO adult mice. Briefly, mice were euthanized and their legs were removed. Bone marrow precursors were flushed out and cell suspension was further incubated with red blood cells hypotonic lysis buffer. After washing, cells were plated in DMEM media containing 10% FBS supplemented with 20% of L929 supernatant for 7 days for full differentiation of bone marrow‐derived macrophages (BMDMs).

GL261 and BMDMs were co‐cultured in 1 : 1 mix in DMEM medium containing 10% FBS. GL261 cells were plated on top of 1 μm pore size Boyden chambers (Thincert, Greiner, Kremsmünster, Austria), whereas BMDMs were plated on the bottom of the 6‐well plates. The mRNA was isolated from BMDMs at 0, 24 and 48 h using the RNeasy mini kit according to the manufacturer’ instructions (Qiagen, Germantown, MD, USA).