Macrophages were collected as described above and transferred to a V-bottom 96-well plate for staining. The cells were centrifuged at 400 g for 5 min at 4 °C. The supernatant was removed, and the cells were washed once with 200 µl PBA (PBS pH 7.4, 1% w/v BSA (Sigma)).
Fc-receptors were blocked by incubation in PBS supplemented with 10% human pooled serum for 15 min at 4 °C. After washing once more, surface markers and viability were stained in a volume of 50 µl for 30 min at 4 °C, using the antibodies and viability dye described in Supplementary Table3 . Following two washes, the cells were re-suspended in 150 µl PBA and measured on a Cytoflex flow cytometer (Beckman Coulter) or BD FACSVerse system (BD Biosciences). Compensation was performed using VersaComp compensation beads (Beckman Coulter) for single antibody stains; a mixture of live and heat-killed cells was used for single stains of the viability dye (as per the manufacturer’s recommendations). Data analysis was performed in Flowjo (v10.7.1, BD Biosciences) Our gating strategy was as follows: first, a time gate was used if necessary. Then, single cell events were selected using subsequent FSC-A/SSC-A and FSC-A/FSC-H gates. Dead cells were removed from the analysis by selecting the viability dye-negative population. Geometric mean fluorescence intensities were calculated as a measure of surface marker expression.
Fc-receptors were blocked by incubation in PBS supplemented with 10% human pooled serum for 15 min at 4 °C. After washing once more, surface markers and viability were stained in a volume of 50 µl for 30 min at 4 °C, using the antibodies and viability dye described in Supplementary Table