Efferocytosis and Macrophage Activation Assay

BMDMs and PCMs were generated and stimulated as described above. To generate apoptotic cells (ACs), thymii from C57BL/6J mice were harvested and mechanistically dissociated, filtered on 100μm nylons (Falcon), pelleted and resuspended in RPMI medium supplemented with 10% FBS. Apoptosis was induced by UV exposure at 312nm for 10min and cells were maintained in culture for an additional 2 hours. This method results in 70-90% apoptosis^{23 (link)}. ACs were labelled with CellTrace™ Violet Cell Proliferation kit (ThermoFisher) according to the manufacturer’s instructions. Fluorescent ACs were washed twice with PBS before use. For one round efferocytosis: stained ACs were added at a 5:1 ratio on plated macrophages for 45min. For two rounds efferocytosis: unlabelled ACs were added at a 5:1 ratio on plated macrophages for 45min. Cells were then washed 3 times and macrophages were incubated for 1h. Stained ACs were then added at a 5:1 ratio on macrophages for 45min. Cells were washed 3 times and macrophages were stained and analysed for AC content and activation markers by flow cytometry. In some experiment, macrophages were treated for 15 min prior efferocytosis with cytochalasin D (5μM, Sigma). For Seahorse extracellular flux analysis ACs were injected directly, before drug treatment, during the assay.