Hardset mountant with 4 6 diamidino 2 phenylindole dapi

Cells were incubated with goat F(ab’)₂ anti-human IgM-coated or control antibody-coated latex beads at a 2:1 bead to cell ratio for 3 h at 37 or 4°C. Cells were then placed on ice for 10 min to terminate internalization. For confocal analysis, 2 × 10⁶ cells were incubated in 1× CellMask™ Deep Red solution (ThermoFisher) as per manufacturer’s instructions for 10 min at 37°C, placed on ice and fixed for 15 min using 4% paraformaldehyde. Samples were then washed and incubated with AlexaFluor488-conjugated donkey anti-goat IgG antibody (20 μg/mL; ThermoFisher A-11055) for 30 min on ice. Cells were washed and cytospun (Thermo Shandon Cytocentrifuge, C4) onto glass slides, dried for 10 min at room temperature and mounted using Vectashield Hardset mountant with 4’,6-diamidino-2-phenylindole (DAPI: Vector Laboratories, H-1500). Images were collected on a Leica SP5 CLSM with a 100× (NA1.4) Plan-apochromatic objective using LAS-AF software (Version2, Leica) and processed using Leica Application Suite X (Leica Microsystems). For flow cytometry, 1 × 10⁶ cells were stained with the AlexaFluor488-conjugated donkey anti-goat IgG antibody (20 μg/mL) for 30 min, washed and then analyzed.