Phagocytosis and Efferocytosis Assays

For determining phagocytic capacity of macrophages, pHRodo Red E.coli BioParticles Conjugate for Phagocytosis kit (ThermoFisher) was used at a concentration of 1 mg/mL according to the manufacturer’s instructions. The pHRodo Red conjugate is non-fluorescent outside the cells but will be brightly fluorescent in the low pH environment of phagosomes. Briefly, isolated macrophages pooled from the exocrine and endocrine fractions of four pancreata were cultured for 30 min with pHRodo E.coli BioParticles at 37°C. For determining efferocytic capacity of macrophages, isolated macrophages were cultured for 30 min with tdTomato-fluorescent apoptotic neutrophils (from Catchup mice) at 37°C in a 1:30 ratio (each tube contained approximately 2,000 macrophages and 60,000 neutrophils). These experiments were performed in 1 mL complete culture medium (RPMI1640 supplemented with 10% FCS and penicillin/streptomycin). Following phagocytosis and efferocytosis assays, cell suspensions were stained with antibodies against CD45 and F4/80 ( Supplemental Table 1 ). Cells were analyzed using a CytoFlex flow cytometer with CytExpert software (Beckman Coulter).