Efferocytosis Assay with BMDMs

Efferocytosis assays were performed using BMDMs as described previously.^{34 (link)} Following treatment of BMDMs with or without 5 μM staurosporine (to induce apoptosis) or LPS (200 ng/ml)+100 μM zVAD (to induce necroptosis) for 24 h, Vybrant dye (5 μM; Invitrogen, Waltham, MA, USA) was added for 30 min at 37 °C to label the cells. Labeled cells then were added to monolayers of naïve unlabeled BMDMs at a ratio of 2:1. After 2 h incubation, efferocytosis of the labeled cells by naïve cells was calculated as a proportion of uptake of labeled cells by quantifying fluorescence intensity using a plate reader.