RAW264.7 phagocytosis assay

RAW264.7 macrophage are an established model to study ATG16L1 during LAP (Lystad et al., 2019). IgG-coated latex beads were prepared as previously described (Jacquin et al., 2019). Briefly, 3-micron beads (Polysciences Inc) were resuspended in 0.1 M Borate and incubated with human IgG at 4°C overnight while rotating. The beads were washed in PBS x3, then resuspended in PBS. Opsinized zymosan was prepared by mixing zymosan with human serum for 30 mins at 37°C followed by washing and resuspension in PBS. RAW264.7 cells were seeded in 15 cm² dishes and treated with 200 U/ml IFNγ (Peprotech, 315-05) for 24 hours prior to use. Where indicated, 350 ul IgG beads, or 175 ul zymosan (10mg/ml), were added to dishes for 30 minutes at 37°C. Cells were washed in cold PBS x 1 and lysed in 900 ul lysis buffer consisting of: 50 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP40 (IGEPAL CA-630, Sigma I3021), phosphatase inhibitors (1x, Sigma P0044) and protease inhibitors (1x, Sigma P8340). Samples were scraped into pre-chilled 1.5 mL Eppendorf tubes, incubated on ice for 20 minutes and centrifuged at 13,500 rpm for 10 minutes at 4°C. Notably, induction of LAP was so robust and specific under these conditions, that phagosome enrichment was not necessary. The supernatants were subjected directly to GFP-TRAP IP, as described below.