Anti mouse cd47 mab clone miap410

Spleen cells were harvested from WT and JAK2 mutant mice and macrophages were generated by culturing the cells in IMDM (Invitrogen) with 10% FBS and murine M-CSF (PeproTech) for 6 days. Macrophages were detached and plated in a 24-well plate with 2.5 × 10⁵ cells per well with 1 ml medium 1 day prior to the assay. The following day, RBCs from PV mice were incubated with CellTrace™ Yellow (Invitrogen) according to the manufacturers protocol. The RBCs were added to the macrophages at an effector to target ratio of 1:10 and the anti-mouse CD47 mAb (clone MIAP410; BioXCell) or a mouse IgG1 isotype control (clone MOPC-21; BioXCell) were added at a concentration of 5 μg/ml. The macrophages and RBCs were co-incubated for 2 h, then washed, stained, and analyzed. Phagocytosis of RBCs was defined by flow cytometry as the percentage of CellTrace™ Yellow-labeled cells out of F4/80+Aqua- cells.

Five weeks post-transplantation, mouse RBCs from WT and JAK2 mutant mice were labeled by intravenous injection of 1 mg EZ-Link™ Sulfo-NHS-Biotin (Thermo Fischer). Six days after biotin injection, mice were treated intraperitoneally 3 times a week for 2 weeks with an anti-mouse CD47 mAb (clone MIAP410; BioXCell) or a mouse IgG1 isotype control (clone MOPC-21; BioXCell) at a dose of 200 μg/mouse. For RBC cell half-life measurement, PB was obtained by sublingual vein bleeding at 48 h post-injection and then twice weekly for up to 5 weeks. For analysis, PB was stained with the following antibodies: anti-mouse Ter-119 (clone TER-119, eBioscience), Streptavidin (Biolgend), and SYTOX™ Blue Dead Cell Stain (Invitrogen). Stained cells were analyzed using the BD LSRFortessaTM cell analyzer.