Efferocytosis Assay

The differentiated BMDMs were plated on 6-well plate starved in DMEM/F12K medium with 0.5% FBS with or without dexamethasone for 18 hrs. The apoptotic cells were prepared from CEM, human T lymphoblast, cells by UV irradiation (50000 μJ/cm²) using CL-1000 UV cross linker. The irradiated CEM cells were incubated for 5 hrs in RPMI medium only in the 37 C incubator followed by staining with 100 ng/ml pHrodo-SE (Invitrogen) for 30 min. The labeled cells were then washed twice in PBS containing 1% BSA and 1 mM EDTA and once with DMEM/F12 medium only. The apoptotic cells were then incubated for 10 min with Gas6 supernatant. The apoptotic cells-Gas6 mixture was then added to macrophages at the ratio 3:1 (apoptotic cells: macrophage) and incubated for 45 min at 37°C. The macrophages were washed twice with PBS and then scraped using cell scraper. Efferocytosis was assessed by analyzing CD11b, F4/80 and pHrodo positive macrophages using flow cytometry.

For ex vivo efferocytosis, peritoneal macrophages were incubated with various concentrations of either the anti-Mertk or Isotype control antibodies. Thymocytes were collected and treated with camptothecin for 2 hours to induce apoptosis, washed and then treated with pHrodo for 30 minutes at room temperature and washed. Labeled thymoctes (5×10⁵) were then added to the peritoneal macrophages and allowed to incubate for 1 hour. Cells were then harevsted and stained with CD11b and F4/80 antibodies (BioLegend) run on a BD FACSCanto. The proportion of pHrodo positive CD11b+F4/80+ cells was then determined and IC₅₀ determinations made using XLFit. For in vivo efferocytosis, 1 ml PBS was injected intraperitoneally in WT and Mertk^(−/−) mice 48 hrs before apoptotic cells injection. Apoptotic cells were prepared and stained as mentioned earlier and 1 X 10⁷ pHrodo stained apoptotic cells were injected in the intraperitoneal cavity for 1 hr. Peritoneal exudate was collected and macrophage efferocytosis was analyzed by flow cytometry.