1. Preparation of poly-L-lysine coverslips

1. Before the B cell activation assay with Ag-coated beads, prepare poly-L-lysine-coated coverslips (PLL-coverslips). Use a 50 mL tube containing 40 mL of 0.01% w/v of PLL solution and immerse the 12 mm-diameter coverslips into the solution. Rotate overnight at RT.
2. Wash the coverslips with 1xPBS and leave to dry on a 24-well plate lid covered with paraffin film. Proceed with B cell activation.

2. B cell activation on antigen-coated coverslips

1. Preparation of antigen-coated coverslips
   1. Before activation, prepare the antigen solution (1x PBS containing 10 µg/mL BCR-ligand+ and 0.5 µg/mL rat anti-mouse CD45R/B220).
   2. NOTE: Consider preparing the coverslips the day before the assay. B220 improves B cell adhesion, however, the BCR-Ligand+ is sufficient to generate the spreading response.
   3. Place the 12 mm coverslip onto a 24-well plate lid covered with paraffin film, add 40 µL of antigen solution onto each coverslip, and incubate at 4 °C overnight. Seal the plate to avoid evaporation of antigen solution.
   4. Wash the coverslips with 1x PBS and air dry.
2. B cell activation
   1. To start the activation, dilute IIA1.6 B cells to 1.5 x 10⁶ cells/mL in CLICK medium + 5% FBS.
   2. Add 100 µL of cells onto an antigen-coated coverslip (Ag-coverslip) and activate for different time points in a cell incubator at 37 °C / 5% CO₂. The typical activating time points are 0, 30, and 60 min.
      NOTE: As in section 1, we recommend starting with the longest activating time point in order to fix all the samples at the same time.
   3. For time 0, place the 24-well plate lid containing the Ag-coverslip on ice and add the cells. Incubate for 5 min on ice.
   4. Carefully aspirate the media on each Ag-coverslip and then add 100 µL of cold 1x PBS to stop the activation.