Macrophage Isolation and Stimulation Protocol

Macrophages were prepared from adult male C57BL/6 (WT) mice (Harlan) and P2X7R KO mice [60 (link)] as described previously [61 (link)]. In brief, the peritoneal cavity was lavaged with RPMI 1640 media (Sigma). Cells were collected by centrifugation (250 x g, 5 minutes) and plated in 24-well plates at a density of 5 x 10⁵ cells/well in RPMI 1640 media (Sigma) supplemented with 5% FBS (PAA Laboratories), 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma). Cells were cultured overnight (37 °C, 5% CO₂) before non-attached cells were removed by a media change. Cells were incubated with LPS (1 μg/ml for 2 hours) before treatment with LL-37 and ATP as indicated.