Mouse BMDM differentiation and culture

Hind legs were collected from C57/B6J mice (aged 6–15 weeks) and cleaned using a scalpel, followed by flushing of the bone marrow with a 1 mL syringe, phosphate-buffered saline (PBS) and 25 G needle. Red blood cells were lysed using ACK lysis buffer following the manufacturer’s instructions (10× red blood cell lysis buffer, 420301; BioLegend). Bone marrow cells were cultured on non-tissue culture-treated 10-cm Petri dishes in complete Iscove’s modified Dulbecco’s media (IMDM), 10% FBS, 50 µM β-mercaptoethanol and supplemented with 25 ng/mL of macrophage colony-stimulating factor (315-02; Peprotech). BMDMs were supplemented with macrophage colony-stimulating factor every 3 days until day 7 of differentiation. BMDMs were activated with 1 ng/mL of LPS purchased from Sigma (E. coli O111:B4 LPS25) for either 5 or 18 h according to experiment type. Further experiments were performed with the glutaminase inhibitor CB-839 (Merck, AMBH2D6FB23B) (1 µM per well) or rapamycin (R8781; Sigma) (50 nM per well) with their respective vehicle controls.