*2.6. Phagocytosis Assay*

Mpcs were labelled using the CellVue Claret Far Red kit (Sigma-Alrich MinClaret) by following the manufacturer's instructions (Sigma-Aldrich) and treated with staurosporin at 5 μM for 4 h, in order to induce apoptosis. M1 and M2c polarized MPs were incubated with apoptotic mpcs at a 1:3 ratio for 30 min at 4 ◦C or 6 h or 16 h at 37 ◦C. After three PBS washings, MPs were detached using trypsin and a cell scraper and cells were labelled with a CD64-APC (BD Pharmingen 558539) and analyzed by flow cytometry using a FACS Aria III cell sorter (BD Biosciences). The double-positive cells (CD64+/Far Red<sup>+</sup> cells) were phagocytic MPs, whereas the CD64+/Far Red<sup>−</sup> cells were nonphagocytic MPs. To exclude MPs that have bound, but not ingested, apoptotic cells, we subtracted the percentage of double-positive cells observed at 4 ◦C from the value observed at 37 ◦C. In some experiments, MPs were treated with 1 μg/mL of cytochalasin D (Sigma-Aldrich C8273), 45 min before adding apoptotic mpcs, and with the added mpcs.
