α igm

Hyaluronic acid (Sigma) or α-IgM (Zymed) coated 96 well polystyrene plates (Nalge Nunc International) were incubated for 1 h at 37°C. The plates were then washed twice with PBS before adding 4 × 10⁵ panning-enriched B cells in 200 µl of RPMI 1640 per well. The cells were adhered for 1 h at 37°C. The plates were then washed with 300 µl of PBS, fixed with 4% paraformaldehyde for 10 min, before adding crystal-violet (7.5 g/l crystal-violet, 2.5 g/l NaCl, 1.57% formaldehyde, 50% methanol) for an additional 5 min. The cells were washed extensively eight times with distilled water, solubilized with 10% SDS, and the amount of dye remaining in the plates was recorded at 540 nm (Multiskan Ascent, Thermo Scientific, Waltham, MA, USA). After subtraction of the non-specific dye bound to empty wells, absolute binding was calculated. The absorbance was determined in at least four wells per condition, in three independent experiments.

Primary enriched B cells were treated with NIM-R8, α-IgM (Zymed), α-transferrin receptor (TfR) (Santa Cruz Biotechnology) or HA (Sigma), and incubated for 15 min at 4°C. The cells were then washed with cold PBS and α-Rat-PE or α-Goat-PE, incubating for 60 min at 37°C to induce cross-linking with the antibodies. To stimulate crosslinking with HA, the cells were incubated for 30 min at 37°C with HA-FITC. Finally, the cells were fixed with 4% PFA and permeabilized with 0.1% of Triton X-100 to detect Myo1g, caveolin, or GM1 as described below. After staining, the cells were mounted on coverslips treated with poly-l-lysine (Sigma) for microscopy observations. In some experiments, cholesterol was removed in B lymphocytes. Briefly, cells were washed twice with 1× PBS and treated with 5 mM of MβCD (Sigma) for 15 min at 37°C (15 (link), 17 (link)) or treated with MβCD plus cholesterol or lanosterol at 1 µg/ml for 30 min at 37°C. To block the recycling of adhesion molecules, the cells were washed twice with 1× PBS and treated with 50 µM of chloroquine (16 (link)), 0.3 mM of primaquine (39 (link)), or 50 µM of Golgi-stop (monensin) (40 (link)) for 1 h at 37°C.