Deepred dye

Apoptotic thymocytes were stained with 2.5 µM DeepRed dye (Invitrogen, Carlsbad, CA, USA) for 24 h according to the protocol provided by the manufacturer, while BMDMs were stained with 10μM carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Thermo Fisher Scientific, Waltham, MA, USA). Apoptotic thymocytes were added to 2 × 10⁵ C57BL/6 macrophages in 1:5 macrophage:target cell ratio for 1 h, then the remaining cells were washed away. BMDMs were then fixed with 1% paraformaldehyde. Pictures were then taken on a fluorescent microscope (FLoid™ Cell Imaging Station, ThermoFisher, Waltham, MA, USA.

BMDMs or peritoneal macrophages were stained for 24 h with 10 μM 5(6)-carboxyfluorescein diacetate (CFDA-SE) (Invitrogen, Carlsbad, CA, USA). To generate apoptotic thymocytes, the thymus was collected from 4 week old C57BL/6 mice, thymocytes were isolated and cultured for 24 h (10⁷ cells/mL) in RPMI 1640 medium supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2.5 μM Deep Red dye (Invitrogen, Carlsbad, CA, USA) in the absence of serum. The percentage of apoptotic thymocytes determined by annexin V labeling was ≥ 80%. Stained apoptotic thymocytes were added to the macrophages in a 5:1 (apoptotic cells:macrophage) ratio for 1 h. After coculture, apoptotic cells were washed away and macrophages were detached by trypsinization. For long-term phagocytosis assays, macrophages were first exposed to unstained apoptotic cells for 5 h and then to the stained apoptotic cells for an additional hour. Cells were analyzed using either FACSCalibur or confocal microscopy.