Anti cd47 clone b6h12

For all flow-based in vitro phagocytosis assays, tumor cells and human macrophages were co-cultured at a ratio of 2:1 in ultra-low-attachment 96-well U-bottom plates (Corning) in serum-free RPMI (Thermo Fisher Scientific). Tumor cells either virally expressed GFP or were labeled with CFSE (Invitrogen) by suspending cells in PBS (5 μM working solution) as per manufacturer instructions for 20 min at 37 °C protected from light and washed twice with 20 ml of FBS-containing media before co-culture. Anti-GD2 (dinutuximab, acquired from United Therapeutics) or anti-B7-H3 (enoblituzumab, acquired from MacroGenics) was added alone or in combination with anti-CD47 (clone B6H12, Bio X Cell) at a concentration of 10 μg ml⁻¹ (or the concentration indicated in the figures). Tumor cells and antibodies were incubated for 30 min in a humidified 5% CO₂ incubator at 37 °C. Plates were washed two times; human macrophages were added to the plate; and plates were incubated for 1–2 h at 37 °C. Phagocytosis was stopped by washing with 4 °C PBS and centrifugation at 336g before the cells were stained with Live/Dead stain and anti-CD11b. Assays were analyzed by flow cytometry, and phagocytosis was measured as the number of CD11b⁺ and CFSE/GFP⁺ macrophages, quantified as a percentage of the total CD11b⁺ macrophages and normalized to the control condition.

Ramos cells were analysed by flow cytometry using the following antibodies: anti-CD20-APC (clone 2H7, Biolegend), anti-CD2-APC (clone REA972, Mitenyi Biotech), anti-CD47 (clone B6.H12, BioXCell), and anti-Calreticulin-DyLight-488 (FMC 75, Enzo Life Sciences), and with Annexin-V FITC (A13199, Thermo Fisher), or with biotinylated Maackia amurensis lectin II (MAL-II) (B-1265, VectorLabs) followed by streptavidin-APC (Thermo Fisher). Sialidase (neuraminidase from Vibrio cholerae, Sigma 11080725001) treatment was performed by incubating cells with 30 nM enzyme in dPBS at a cell concentration of 1 × 10⁶ ml⁻¹ for 1 h at 37 °C. In Extended Data Fig. 6j, single-cell suspensions were prepared from diced tumours and fixed in paraformaldehyde as described previously. Cells were stained with Zombie-NIR viability dye (BioLegend), anti-CD45 (clone 30-F11, BioLegend), anti-F4/80 (clone BM8, BioLegend) and anti-CD11b (clone M1/70, BioLegend) and analysed using an Acea Novocyte Quanteon flow cytometer and FlowJo software (version 10.6.1). Live CD45⁺ cells were counted using gates that separated the CD45⁻ and CD45⁺ populations, and macrophages were counted using a single gate applied equally to all samples.