In vitro efferocytosis assay

To examine Mϕ efferocytosis in vitro, we adapted an established protocol^17–19. Briefly, neonatal cardiomyocytes (or thioglycollate-elicited peritoneal neutrophils) were labeled with DiI (Invitrogen), UV-irradiated (254nm, 7 minutes), and then incubated at 37°C for 2 hours. The resulting apoptotic cells were collected and added to Mϕ cultures. At the appropriate time point (20 or 60 minutes), cells were washed and then collected for flow cytometry or immunocytochemical analyses. For the inhibitor study, anti-IgG or anti-C1qa (Abcam) were added in conjunction with apoptotic cells.