B Cell Activation on an Antigen-Coated Coverslip

Abstract

Source: Ibañez-Vega, J., et al., Studying Organelle Dynamics in B Cells During Immune Synapse Formation. J. Vis. Exp. (2019)

This video demonstrates B cell activation in vitro. The process involves antigen interaction with B cell receptors, leading to cytoskeleton remodeling, immune synapse formation, lysosome movement, antigen internalization, processing, and subsequent presentation on MHC molecules for B cell activation.

Protocol

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IIA1.6 (A20 variant) mouse B-lymphoma cells	ATCC	TIB-208	Murine B-cell lymphoma of Balb/c origin that expresses an IgG-containing BCR on its surface without FcγIIR
CaCl2	Winkler	CA-0520
Glycine	Winkler	BM-0820
HyClone Fetal bovine serum	Thermo Fisher Scientific	SH30071.03	Heat inactivate at 56 C for 30 min
KCl	Winkler	PO-1260
NaCl	Winkler	SO-1455
Parafilm	M	P1150-2
Penicillin-Streptomycin	Thermo Fisher Scientific	15140122	Liquid
Poly-L-Lysine	Sigma	P8920	Dilute with sterile water
RPMI-1640	Biological Industries	01-104-1A
Sodium pyruvate	Thermo Fisher Scientific	11360070
Tube 50 ml	Corning	353043
2-mercaptoethanol	Thermo Fisher Scientific	21985023
Glutamine	Thermo Fisher Scientific	35050061
Goat-anti-mouse IgG antibody	Jackson ImmunoResearch	315-005-003	IIA1.6 positive ligand