Isolation of Murine Resident Peritoneal Cells

Murine RPCs were isolated from the peritoneal lavage fluid of naive mice as previously described (2 (link), 4 (link)). Naive mice (n = 40) were anesthetized using CO₂ and sacrificed by cervical dislocation. The abdominal cavity was flushed with 5 mL Ca²⁺/Mg²⁺-free DPBS (Gibco, Waltham, MA, USA). The collected peritoneal lavage fluid was centrifuged at 400 g for 10 min at 4°C, and the pelleted RPCs were resuspended in complete RPMI-1640 (Gibco) medium [RPMI-1640 medium containing 0.05 mM 2-β-mercaptoethanol (Sigma-Aldrich), 3 mM L-glutamine (Sigma-Aldrich), 10 mM HEPES (Sigma-Aldrich), 100 U/mL penicillin/streptomycin (Sigma-Aldrich), and 10% fetal calf serum (HyClone)] and incubated at 37°C with 5% CO₂ (4 (link)). All cells were freshly isolated prior to use.