This article discusses the activation of B cells through antigen recognition by B cell receptors (BCRs). It details an in vitro method for studying B cell activation using coated coverslips and B cell incubation.
B cell activation involves the recognition of antigens by B cell receptors, BCRs, on their surface to elicit an immune response.
To study B cell activation in vitro, begin with a coverslip containing antigens and anti-CD45R antibodies, immobilized over a polymeric material. The negatively-charged antigens and antibodies interact with positively-charged polymers on the coverslip through electrostatic interactions.
Overlay the coated coverslips with a medium containing B cells and incubate. The CD45R — the transmembrane protein of B cells — interacts with the immobilized antibodies, facilitating cell adhesion.
The BCRs on the adhered B cell interact with an antigen, forming an immune synapse — the coordination region for cell signaling and antigen internalization. This promotes cytoskeleton remodeling and initiates cell spreading, which increases the cell surface in contact with the antigens.
Further remodeling causes the movement of BCR-coupled antigens toward the center of the immune synapse. This cellular rearrangement causes the repositioning of the centrosome near the synaptic membrane that guides intracellular organelles, including lysosomes, toward the synapse.
The fusion of lysosomes with the membrane leads to the internalization of the antigen-BCR complexes. The internalized antigens are processed into peptide fragments, loaded onto class-II major histocompatibility complex or MHC molecules, and finally presented on activated B cells.
Prepare the antigen solution by adding anti-BCR and B220 in PBS to a final concentration of 10 micrograms per mL and 7.5 micrograms per mL respectively. Next, set the coverslip over a lid of 24-well plate covered with paraffin film, and add 40 microliters of antigen solution. Seal the plate, and then incubate at four degrees overnight.
To start the activation in coverslip, first, wash them with PBS, and let them dry for a few minutes. Next, add 150,000 B cells, and incubate at 37 degrees for the different time points. In both cases, when activating either with beads or antigen-coated slides, we recommend starting with the longest time point, and then proceeding to the shorter ones.
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