RAW264.7 Macrophage Cell Culture

The RAW264.7 macrophage cell line was sourced from the American Type Culture Collection (ATCC), located in Manassas, VA, USA. These cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM, Gibco brand) which was fortified with 10% fetal bovine serum (Gibco) and an additional 1% penicillin/streptomycin antibiotic blend (Gibco). The culture conditions maintained were at a steady temperature of 37°C under a humidified atmosphere containing 5% CO₂. RAW264.7 cells were seeded onto 10 cm cell culture dishes (Corning Inc., New York, USA) and the medium was refreshed every two days to ensure optimal growth. Cell passage was carried out once the cells achieved approximately 90% confluence. This process involved discarding the used culture medium, followed by rinsing the adherent cells twice with PBS. Subsequently, the cells were detached and re-suspended in fresh medium at a ratio of 1:3 for further propagation. In subsequent experiments, RAW264.7 cells were treated with 1 μg/ml LPS (L3024, Sigma, MO, USA) for 24 h as described in our previous publication [59 (link)]. Erastin (10 μM) (Selleck, USA), rapamycin (20 μM) (Selleck), 4-P-PDOT (10 μM) (Sigma) and luzindole (10 μM) (Sigma) were treated 3 h before LPS stimulation. MT (500 μM) (Sigma) was treated 1 h after LPS stimulation. All cells were verified to be free of mycoplasma.