Scanning electron microscopy (SEM)
For SEM experiments, antigen coated coverslips were used. Sterile coverslip were coated in 24-well plates with 60 μl poly-D-lysine solution (50 μg/ml) (C0312, Beyotime) overnight at 4 °C. The next day, the coverslips were washed with sterile PBS, dried, and incubated with 100 μl 10 μg/ml F(ab’)2 antibody at 37 °C for 3 h. Purified B cells at a concentration of 3 × 106 cells/ml were added gently onto the antigen coated coverslips and stimulated at 37 °C for 10 min. A total of 400 μl of 2.5% glutaraldehyde was used to terminate the stimulation and fix the cells for 20 min (on ice). Scanning electron micrographs were captured using a Ultra-high Resolution Scanning Electron Microscope (SU8010, HITACHI, Tokyo, Japan). Cellular filopodia expansion was imaged and the number and length of filopodia were quantified using ImageJ software (NIH, Bethesda, MD, USA).