Efferocytosis assays

To directly assess efferocytosis, apoptotic thymocytes and T cells were first stained with CFSE and then used to feed BMDMs or peritoneal macrophages. To obtain apoptotic cells for these assays, T cells from naïve or infected B6 mice were first cultured for 24 h in DMEM complete medium at 2 × 10⁶ cells/mL in 24-well plates. Thymocytes from normal B6 thymuses were cultured at 2 × 10⁶/mL in supplemented RPMI medium (24-well plates) for 72 h to obtain apoptotic thymocytes⁶⁶. T cells and apoptotic thymocytes were then labelled with 0.5 µM CFSE. BMDMs and peritoneal macrophages from T. cruzi-infected mice were cultured at 1 × 10⁶ cells/well for 24 h and then incubated with 5 × 10⁵ CFSE-labelled thymocytes or T cells. After 1 h, nonadherent cells were washed out, and macrophages were collected and stained with anti-CD8/anti-TCRβ and anti-CD11b/anti-F4/80 for flow cytometry analyses. After excluding doublets and TCRβ⁺ cells, CD11b⁺CFSE⁺ cells correspond to macrophages that have phagocytosed (intracellular) CFSE⁺ cells. Gates were set based on the analyses of macrophages cultured alone or with CFSE-negative cells. Otherwise, gated F4/80⁺ cells were analysed for CD8⁺CFSE⁺ events for the identification of extracellular thymocytes.