In Vitro Phagocytosis Assay for Macrophages

The in vitro phagocytosis assay was performed as described before²⁴ (link)^,27 (link), with slight modifications. Buffy coats and human serum were obtained from the Swiss Blood Bank (Interregionale Blutspende SRK, Bern, Switzerland). PBMCs were enriched from buffy coats by density centrifugation using Lympho Spin Medium (pluriSelect). Monocytes were isolated from PBMCs using the EasySep Human CD14 Positive Selection Kit II (Stemcell Technologies) according to the manufacturer's instructions. 4–5 × 10⁶ monocytes per well were differentiated into macrophages for 7 days in 6-well tissue culture plates in Iscove's modified Dulbecco's medium supplemented with 10% human serum, L-glutamine and penicillin/streptomycin. ACC-MESO-1 cells were harvested using non-enzymatic cell dissociation buffer (Sigma) and labelled with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) in PBS at a final concentration of 20 µM. Macrophages were starved in serum-free medium for 2 h and 1 × 10⁶ CFSE-labelled ACC-MESO-1 cells were added. Cell were co-cultured for 2 h in the presence of 10 µg/ml mouse anti-human CD47 (clone B6H12.2, ThermoFisher) or isotype control (mouse IgG1, ThermoFisher), then harvested, stained for CD45 and CD14 (Table S1) and analysed by FACS.