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 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. 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…
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
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Cite This Article
B Cell Activation on an Antigen-Coated Coverslip. J. Vis. Exp. (Pending Publication), e21611, doi: (2023).