Cell proliferation dye v450

Macrophages were harvested and pre-seeded at 1.5 × 10⁴ cells/well in 96-well plates. Tumor cells were labeled with cell proliferation dye V450 (Thermofisher) or pHrodo green pH indicator (Thermofisher) according to manufacturer’s instructions. Subsequently, tumor cells were incubated with or without anti-human CD47 IgG4 antibody (Inhibrix) or F(ab′)2 of Inhibrix (both at 5 μg/ml) on ice for 1 h. Tumor cells were washed with cold PBS (two times) and added to pre-seeded macrophages (effector to target ratio of 1:5) and incubated for 3 h at 37 °C. Tumor cells were gently removed by washing with PBS 2–3 times and phagocytosis was analyzed by fluorescent microscopy (Leica, DM6000) or confocal microscopy (Leica SP8). For visualization of macrophages, residual cells were stained with anti-human CD11b-alexa594 (1 μg/ml) at RT for 45 min. The percentage of phagocytosis was calculated by counting the number of macrophages containing V450-labeled tumor cells per 100 macrophages. Each condition was quantified by evaluating three randomly chosen fields of view.

PE-labeled anti-human SLAMF7 antibody (clone, 162.1) and PE-labeled isotype control were purchased from Biolegend. APC-labeled anti-CD3, FITC-labeled anti-CD19 and APC-labeled anti-CD47 were purchased from Immunotools (Germany). FITC-labeled anti-CD20 was purchased from Thermofisher. Alexa594-labeled CD11b was purchased from Biolegend. Anti-human CD47 IgG4 antibody (Inhibrix, clone B6H12) was generated in-house by Aduro Biotech Europe (ABE). F(ab′)2 of Inhibrix was prepared with Pierce F(ab′)2 preparation kit. F(ab′)2 generation was confirmed by staining for human IgG4 (Supplementary Figure 1D). Cell proliferation dye V450 was purchased from Thermofisher. Phrodo Green pH indicators was purchased from Thermofisher. Lymphoprep was purchased from Axis-Shield PoC AS, Norway. pHrodo Green intracellular pH indicator was purchased from ThermoFisher. RNeasy mini kit was purchased from Qiagen. iScript cDNA Synthesis Kit was purchased from Bio-Rad.