Peripheral blood and splenocytes were stained with Gr1(Ly6C/G)-PerCP-Cy5.5 (BD Pharmingen, cat# 552093), CD115-APC (eBioscience, cat# 17-1152-82), CD11b-APC Cy7 (BD Bioscience, cat# 557657), and V450-CD45 (BD Horizon, cat# 560501) (30 (link)). Data were acquired on a BD FACS Canto II instrument (BD Biosciences) and analyzed using FACSDiva software v6.1.3 (BD Biosciences).
To examine the apoptotic rate, Jurkat T cells were stained with Annexin V-APC and PI (Thermo Fisher). To examine efferocytosis, Jurkat T cells were first stained with cell tracker green CMFDA (2 μM) at 37°C for 30 min before treated with 1 μM staurosporine for 4 h to induce apoptosis. Fluorescent labeled apoptotic Jurkat T cells were then incubated with macrophages at 37°C for 0-120 min. Macrophages were then washed with PBS for five times and incubated with accutase dissociation buffer at 37°C for 30 min. Data were acquired on a BD FACSCalibur (BD Biosciences) and analyzed using Flowjo V7.6.5 (BD Biosciences).
To examine the apoptotic rate, Jurkat T cells were stained with Annexin V-APC and PI (Thermo Fisher). To examine efferocytosis, Jurkat T cells were first stained with cell tracker green CMFDA (2 μM) at 37°C for 30 min before treated with 1 μM staurosporine for 4 h to induce apoptosis. Fluorescent labeled apoptotic Jurkat T cells were then incubated with macrophages at 37°C for 0-120 min. Macrophages were then washed with PBS for five times and incubated with accutase dissociation buffer at 37°C for 30 min. Data were acquired on a BD FACSCalibur (BD Biosciences) and analyzed using Flowjo V7.6.5 (BD Biosciences).