Organelle Dynamics in B Lymphocytes following Activation with Antigen-Coated Beads

NOTE: The following steps were performed using IIA1.6 B cells.

1. B cell activation with antigen-coated beads

1. Preparation of antigen-coated beads
   1. To activate B cells, use NH₂-beads covalently coated with antigen (Ag-coated beads), which are prepared using 50 µL (~20 x 10⁶ beads) of 3 µm NH₂-beads with activating (BCR-ligand+) or non-activating (BCR-ligand-) antigens.
   2. For IIA1.6 B cells use anti-IgG-F(ab')₂ fragment as BCR-ligand+ and anti-IgM-F(ab')₂ or bovine serum albumin (BSA) as BCR-ligand-. To activate primary B cells or IgM+ B cell line use anti-IgM-F(ab')₂ as BCR-ligand+ and BSA as BCR-ligand-.
      NOTE: To avoid the binding of ligands to the Fc receptor, use F(ab') or F(ab')₂ antibody fragments instead of full-length antibodies.
   3. To proceed with the preparation of Ag-coated beads, place the beads in low protein binding microcentrifuge tubes to maximize the beads' recovery during the sample manipulation. Add 1 mL of 1x phosphate-buffered saline (PBS) to wash beads and centrifuge at 16,000 x g for 5 min. Aspirate the supernatant.
   4. Resuspend the beads with 500 µL of 8% glutaraldehyde to activate the NH₂ groups and rotate for 4 h at room temperature (RT).
      CAUTION: The glutaraldehyde stock solution should only be used in a chemical fume hood. Follow the instructions on the material safety data sheet (MSDS).
   5. Centrifuge beads at 16,000 x g for 5 min, remove the glutaraldehyde, and wash the beads three more times with 1 mL of 1x PBS.
      CAUTION: The glutaraldehyde solution should be discarded as hazardous chemical waste.
   6. Resuspend the activated beads in 100 µL of 1x PBS. The sample can be divided into two low protein binding microcentrifuge tubes: 50 µL for the BCR-ligand+ and 50 µL for BCR-ligand-.
   7. To prepare the antigen solution use two 2 mL low protein binding microcentrifuge tubes containing 100 µg/mL of antigen solution in 150 µL of PBS: One tube with BCR-ligand+ and the other with BCR-ligand-.
   8. Add 50 µL of activated beads solution to each tube containing 150 µL of antigen solution, vortex, and rotate overnight at 4 °C.
   9. Add 500 µL of 10 mg/mL BSA to block the remaining reactive NH₂ groups on the beads and rotate for 1 h at 4 °C.
   10. Centrifuge the beads at 16,000 x g for 5 min at 4 °C and remove the supernatant. Wash the beads with cold 1x PBS three more times.
   11. Resuspend the activated beads in 70 µL of 1x PBS.
   12. To determine the final concentration of Ag-coated beads, dilute a small volume of beads in PBS (1:200) and count using a hemocytometer. Then store at 4 °C until use.
       NOTE: Ag-coated beads should not be stored for more than 1 month.

2. 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 1x PBS and leave to dry on a 24-well plate lid covered with paraffin film. Proceed with B cell activation.

3. B cell activation

1. To start B cell activation with beads, first dilute the IIA1.6 B cell line to 1.5 x 10⁶ cells/mL in CLICK medium (RPMI-1640 supplemented with 2 mM L-Alanine-L-Glutamine + 55 µM beta-mercaptoethanol + 1 mM pyruvate + 100 U/mL Penicillin + 100 µg/mL streptomycin) + 5% heat-inactivated fetal bovine serum [FBS]).
2. Combine 100 µL (150,000 cells) of IIA1.6 B cells with Ag-coated beads at a 1:1 ratio in 0.6 mL tubes. Mix gently using a vortex and seed onto PLL-coverslips. Incubate for different time points in a cell culture incubator (37 °C / 5% CO₂). The typical activating time points are 0, 30, 60, and 120 min. For time 0, place the PLL coverslips into the 24-well plate lid on ice. Add the cells-Ag-coated bead mixture and incubate for 5 min on ice.
   NOTE: It is important to mix by vortex instead of pipetting up and down because this could reduce the number of beads in the sample, due to their accumulation in the plastic tip of the pipette. Use beads coated with BCR-ligand- as a negative control for each assay. We recommend activating the longest incubation time first and then the following time points. Calculate intervals of activation for samples such that they are ready for fixation at the same time.
3. Add 100 µL of cold 1x PBS to each coverslip to stop the activation and continue with the immunofluorescence protocol.