Live dead stain fixable viability dye

Human blood-derived macrophages were generated from anonymous healthy donors as previously described^{68 (link)}. Human blood samples were obtained through the Stanford Blood Center under an Institutional Review Board-exempt protocol. Tumor cells were labeled with CFSE (Invitrogen) as per the manufacturer protocol. Tumor cells were then incubated with the anti-GD2 antibody dinutuximab (obtained from Stanford pharmacy) at a concentration of 10 μg ml⁻¹ or media for 30 min at 37 °C. After incubation, tumor cells were washed twice in PBS and then co-cultured at a 2:1 ratio with macrophages in serum free media for 2 h at 37 °C. After co-culture, cells were washed with PBS and centrifuged at 1,200 × g for 5 min. They were then stained for downstream analysis with anti-CD11b (Clone M1/70, BioLegend) and Live/ Dead stain (Fixable Viability Dye, eBioscience). Flow cytometry was performed on an LRS Fortessa Analyzer (BD Biosciences), and phagocytosis was measured as the percentage of the total CD11b⁺ macrophages that were also CFSE⁺. Phagocytosis in response to dinutuximab was normalized to conditions without antibody.