Evaluating Splenocyte Phagocytosis by Flow Cytometry

After 72 h of incubation, splenocytes exposed to TNP-LPS or not in media supplemented or not with methionine were collected and resuspended in L-15 medium without serum. The cells were then incubated with fluorescent beads (FluoSpheres R Microspheres, 1.0 μm, Crimson Red Fluorescent 625/645, 2% solids; Thermo Fisher Scientific) at a cell:bead ratio of 1:10 as described before for 3 h at 20°C (26 (link)). After the incubation period, cells were harvested by gently pipetting, and non-ingested beads were removed by centrifugation (100 x g for 10 min at 4°C) over a cushion of 3% (weight/volume) BSA (Fraction V; Fisher Scientific) in PBS supplemented with 4.5% (weight/volume) D-glucose (Sigma). Cells were then resuspended in staining buffer, labeled with anti-IgM-FITC (1.14) (1 μg/ml) for 1 h at 4°C in the dark in staining buffer. After this time, cells were washed twice with staining buffer. The cell viability was checked by addition of 4’,6-diamine-2’-phenylindole dihydrochloride (DAPI 0.2 μg/ml). Cells were analyzed on a FACS Celesta™ flow cytometer equipped with BD FACSDiva software. Flow cytometry analysis was performed with FlowJo^® V10.