Annexin 5 recombinant protein

For induction of apoptosis, human Jurkat T cells resuspended in RPMI with 1% BSA were treated with 150 mJ/cm² ultraviolet C irradiation (Stratalinker) and incubated for 4h at 37 °C, 5% CO₂. For antibody-dependent phagocytosis, Jurkat cells were labeled with αCD3 (25μg/ml, Clone: OKT3 BioLegend) along with Annexin V recombinant protein (to block efferocytosis of any residual dying cells) (3μg/ml, eBioscience) for 1h at 4 °C and the cells were then stained with CypHer5E (GE Healthcare, PA15401) or TAMRA (Invitrogen, C-1171) before use in the engulfment assays. Chines hamster LR73 cells or murine macrophages were seeded in a 24 well plate and incubated with targets at a 1:10 phagocyte to target ratio for the indicated times. Targets were then washed with PBS. Where indicated, phagocytes were rested in culture medium for an additional period of time. Cells were dissociated from the plate with trypsin and the phagocytes were assessed by a flow cytometry-based assay or used in their analysis of RNA or protein^{23 ,24} . Phalloidin staining was conducted according to the manufacturer’s instruction (Invitrogen). When primary macrophages were used as phagocyte, there is an inherent difference in the absolute % uptake of corpses between experiments performed on different days. Therefore, phagocytic index was used to better compile data from multiple experiments.