Miap301

SCG/Kj mice were purchased from the National Institute of Biomedical Innovation, Health, and Nutrition (Osaka, Japan). Eight-week-old mice were treated with i.p. injections of either 200 μg of anti-CD47 mAb (MIAP301, BioXcell) or rat IgG2a isotype CT-Ab (2A3, BioXcell) every 5 days for 2 weeks (n = 6 for each). Ten-week-old mice were then analyzed.

C57BL/6J bone marrow was harvested as described above, plated in 100 mm untreated Petri dishes in IMDM (supplemented 10% FBS, 1%P/S, 20 ng/mL M-CSF), and allowed to differentiate into macrophages for 7 days. On day 7 of differentiation, macrophages were washed with 1x PBS, lifted with 0.25% Trypsin for 10 min at 37 °C, and plated in 24- or 6-well plates in the same supplemented IMDM for phagocytosis assays the following day. Targets for phagocytosis were opsonized for up to 30 min at RT or on ice at the following concentrations and antibodies: 10 µg/mL TA99 (BioXCell, Lebanon, NH, USA) for B16F10 variants, and 10 µg/mL anti-streptavidin for polystyrene microparticles. Where applicable against WT targets, mouse targets were CD47-blocked with 10 µg/mL MIAP301 (BioXCell). Prior to adding targets to the wells for phagocytosis, macrophages were labeled with 0.5 µM CellTracker DeepRed in PBS for 15 min and B16F10 cells with 10 µM Vybrant CFDA SE Cell Tracer Kit in PBS for 15 min at RT. Wells were washed with serum-free IMDM (IMDM supplemented with 0.1% BSA, 1% P/S, no M-CSF or FBS) after 2 h. For imaging, wells were fixed for 20 min in 4% paraformaldehyde at RT, then washed with PBS, and stored at 4 °C in PBS prior to imaging.