This article discusses the activation of B lymphocytes through B cell receptors (BCRs) and the subsequent immune response. It details the methodology for studying B cell activation in vitro using antigen-coated beads.
B cell receptors, BCRs, on B lymphocytes, bind antigens, causing cell activation. This leads to antigen internalization for processing into peptide fragments, which are loaded on class II MHC molecules for presentation to helper T lymphocytes to generate an effective immune response.
To study B lymphocyte activation in vitro using antigen-coated beads, first, obtain antigen-coated beads. These beads are covalently coated with antigens, ligands specific for BCRs; a non-reactive protein blocks the unbound bead sites, preventing non-specific B lymphocyte binding.
Add the beads to the B lymphocyte suspension. Seed the mixture onto a poly-L-lysine-coated coverslip.
The negatively-charged B lymphocytes bind to the positively-charged coverslip, immobilizing the cells. Further, the beads' antigens bind specifically to BCRs. The interaction of the BCR with the antigen activates B lymphocytes, forming a tight junction — an immune synapse.
The interaction initiates intracellular signaling pathways, resulting in actin cytoskeleton reorganization. This causes cell membrane spreading followed by contraction, during which the BCR-antigen microcluster concentrates at the immune synapse center.
With actin remodeling, the centrosome — a microtubule organizing center — repositions near the synapse. The repositioning recruits organelles like lysosomes and the Golgi apparatus near the immune synapse to extract and present antigens.
Antigen-coated beads are prepared by incubating specific ligands with amino beads previously treated with glutaraldehyde to activate amino groups. Resuspend activated beads in 100 microliters of PBS and vortex. Meanwhile, prepare the antigen solution by adding 100 micrograms per ml of BCR ligand in 150 microliters of PBS.
Next, add 50 microliters of activated beads to the antigen solution, and incubate overnight at 4 degrees Celsius. Remember, also use a non-related ligand as negative control. After incubation, wash bead with PBS, aspirate the supernatant, and replace with PBS. Repeat three times. Next, block the free amino groups by resuspending beads in 500 microliters of a solution of BSA. Finally, resuspend beads in 70 microliters of PBS.
To calculate the final concentration, you can use a hemocytometer to count the beads. For B-cell activation, use a 50 ml tube and resuspend cells to a concentration of 1.5 million per ml in CLICK supplemented with 5% fetal bovine serum. Next, add 150,000 B-cells corresponding to 100 microliters of the cell-bead mixture to a poly-L-lysine coated coverslip, and incubate at 37 degrees for the different time points.
繁體中文
