Cells were collected by centrifugation at 500 for 5 min, suspended in 300 to 500 l of CHES (for 5 min, suspended in 500 l of CHES-FACS buffer, and analyzed by flow cytometry as described above. cells/well in 96-well microtiter plates. Clones expressing high levels of Tim4 were expanded for further analysis. Flow cytometry. Cells were incubated on ice for 30 min with 1 g/ml hamster anti-mouse Tim4 (clone Kat5-18) (10) and 1 g/ml biotinylated anti-MerTK Ab in a mixture made up of 50 l of PBS and 2% FCS, followed by incubation with 1.0 g/ml Alexa Fluor 488-conjugated streptavidin, 1 g/ml PerCP-Cy 5.5-labeled rat anti-mouse Mac1, and 0.6 g/ml APC-labeled anti-hamster IgG. The cells were then stained with 0.5 M Sytox Blue (Life Technologies) to exclude dead cells and analyzed by flow cytometry with a FACSCanto II instrument (BD Biosciences). Engulfment of apoptotic cells. Engulfment of apoptotic cells was assayed with pHrodo-labeled prey (20, 25). In brief, thymocytes from 4- to 8-week-old C57BL/6J mice were treated with 100 models/ml FasL in Dulbecco’s Rabbit polyclonal to 2 hydroxyacyl CoAlyase1 altered Eagle’s medium (DMEM) made up of 10% FCS for 1.5 to 2 h at 37C to induce apoptosis, washed with PBS, and incubated with 0.1 g/ml pHrodo for 30 min at room temperature. After the reaction was stopped with 1 ml FCS, the cells were washed with PBS made up of 10% FCS and were used as prey. At this stage, the annexin V+ propidium iodide-positive (PI+) cell populace was usually less than 30%. In some cases, thymocytes were incubated with FasL in serum-free DMEM, labeled with pHrodo as described above, and washed with PBS made up of 0.5% bovine serum albumin (BSA) and 0.25% globulin. To prepare peritoneal macrophages, peritoneal cells (5 105) from wild-type and mutant mice at 8 to 14 weeks of age were incubated in 12-well plates at 37C for 2 h in DMEM made up of 10% FCS and were washed with PBS to remove nonadherent cells. The adherent cells were incubated at 37C with 2 106 pHrodo-labeled apoptotic thymocytes in 1 ml of DMEM made up of 10% FCS, washed with PBS, and then treated at 37C with 0.25% trypsinCPBS containing 1 mM EDTA. Cells were collected by centrifugation at 500 for 5 min, suspended in 300 to 500 l of CHES (for 5 min, suspended in 500 l of CHES-FACS buffer, and analyzed by flow cytometry as described above. For microscopic observation, Ba/F3 cells coincubated with pHrodo-labeled thymocytes were suspended in 500 l of CHES-FACS buffer, transferred to Lab-Tek II chambered cover glasses (ThermoFisher Scientific), and examined by fluorescence microscopy (BioRevo BZ-9000; Keyence). Binding of apoptotic cells to phagocytes. The binding of apoptotic cells to 7-xylosyltaxol phagocytes was assayed by using the CellTracker Orange-labeled cells as described previously (20). In brief, approximately 1 108 thymocytes were labeled by incubation in 5 ml of serum-free DMEM made up of 10 M CellTracker Orange at 37C for 30 min and then incubated with FasL in DMEM made up of 10% FCS at 37C for 2 h to induce apoptosis. Peritoneal cells or Ba/F3 cells (1 105) were then coincubated in suspension with the CellTracker Orange-labeled apoptotic cells in PBS supplemented 7-xylosyltaxol with 10% FCS, stained with 500 nM Sytox Blue, and analyzed by FACSCanto II. For peritoneal macrophages, the cells were stained with APC-conjugated anti-Mac1. Immunoprecipitation and Western blotting. Resident peritoneal cells (3 106 to 6 106 cells) on 3.5-cm-diameter plates were incubated at 37C with 1.5 107 to 3 107 apoptotic thymocytes in 1 ml of DMEM made up of 10% FCS, washed with cold PBS to remove apoptotic cells, and lysed by incubation at 4C for 30 min in 1.5 ml of lysis buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EGTA, 1% Triton X-100, 5% glycerol, 2 mM Na2VO4, and a cocktail of protease inhibitors [cOmplete, Mini, EDTA free; Roche]). The lysates were centrifuged at 15,000 rpm for 10 min at 4C, and the supernatants were used for immunoprecipitation. 7-xylosyltaxol Dynabeads protein G (10 l) (Life Technologies) was conjugated with 2.5 g of goat anti-mouse MerTK Ab according to the supplier’s instruction. The macrophage cell lysates prepared from 6 106 cells were incubated overnight at 4C with anti-MerTK-conjugated protein G beads in 1.5 ml lysis buffer. The beads were collected, washed with lysis buffer, and suspended in 30 l of SDS-sample buffer (63 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 0.005% bromophenol blue [BPB], 2% -mercaptoethanol). Proteins were eluted from the beads by heating.