Quantifying Phagocytosis of Apoptotic Cells

Uptake of apoptotic cells by peritoneal MΦres was assessed as previously described (34 (link)). Briefly, thymocytes were collected from naive animals by mincing thymi through 2 μm gauze until completely homogenized. Erythrocytes were removed by incubating with red blood cell lysis buffer (Sigma-Aldrich). Thymocytes were resuspended at 1x10^7 cells/mL in complete DMEM and incubated in the presence of 0.1 µM dexamethasone (Sigma-Aldrich) at 37°C for 18 h. This produced >90% apoptosis, as assessed by Viastain AO/PI staining measured on a Cellometer ^® Auto 2000 Cell Counter (Nexcelom Bioscience). Subsequently, apoptotic thymocytes were washed twice with PBS and resuspended in PBS at 10^6 cells/mL containing 40 ng/mL pHrodo-SE (Thermo Fisher Scientific) and incubated at RT for 30 min. Thereafter the cells were washed twice with PBS and resuspended in RPMI containing 5% foetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. Unstained apoptotic cells served as staining control.