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