WB-F344 and RAW264.7 Co-Culture Protocol

WB-F344 cells (a rat oval cell line that is morphologically and functionally similar to freshly isolated HPCs[23 (link)]) and RAW264.7 cells (a murine macrophage cell line) were purchased from the Shanghai Cell Bank (Chinese Academy of Sciences, Shanghai, China). WB-F344 cells were cultured at 37 °C in an atmosphere containing 5% CO₂ in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mmol/L glutamine, and penicillin/streptomycin (100 mg/mL). Activation of RAW264.7 cells was induced with LPS 100 ng/mL for 8 h at 37 °C in an atmosphere containing 5% CO₂ in DMEM supplemented with 10% FCS[24 (link)], and then co-cultured with WB-F344 cells in Transwell chambers. A total of 2 × 10⁴ WB-F344 cells were seeded into the upper compartment and 4 × 10⁴ RAW264.7 cells were seeded into the lower compartment of the Transwell chamber. LPS was added to the culture medium, and the medium was replaced every 48 h for a total culture time of 7 d.