The endocytosis experiment was performed as described in a previous protocol (16 (link)). Briefly, splenic cDCs were resuspended in prewarmed RPMI 1640 complete medium and incubated with Alexa Fluor 647 (AF647)–conjugated OVA (Molecular Probes O-34784) at a final concentration of 5 μg/ml for 30 min. Uptake of AF647-OVA was terminated by adding ice-cold PBS. The cells were washed three times by cold PBS before flow cytometry analysis.
Latex beads, carboxylate-modified polystyrene, and fluorescent red (L3030, diameter 2 μm; Sigma) were coated for 1 h at 37°C with mouse IgG (Jackson ImmunoResearch). After rinsing and reconstituting in RPMI 1640 complete medium, beads were added to BMDCs at a ratio of 10:1 (beads:BMDCs) and incubated with BMDCs for indicated time periods at 37°C. BMDCs were collected, washed in cold PBS to remove nonadherent beads, and fixed in 4% (w/v) polyoxymethylene. Cells were subjected to flow cytometry analysis.
Latex beads, carboxylate-modified polystyrene, and fluorescent red (L3030, diameter 2 μm; Sigma) were coated for 1 h at 37°C with mouse IgG (Jackson ImmunoResearch). After rinsing and reconstituting in RPMI 1640 complete medium, beads were added to BMDCs at a ratio of 10:1 (beads:BMDCs) and incubated with BMDCs for indicated time periods at 37°C. BMDCs were collected, washed in cold PBS to remove nonadherent beads, and fixed in 4% (w/v) polyoxymethylene. Cells were subjected to flow cytometry analysis.
