Overview
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 activation on antigen-coated coverslips
- Preparation of antigen-coated coverslips
- Before activation, prepare the antigen solution (1x PBS containing 10 µg/mL BCR-ligand+ and 0.5 µg/mL rat anti-mouse CD45R/B220).
- 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.
- 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.
- Wash the coverslips with 1x PBS and air dry.
- B cell activation
- To start the activation, dilute IIA1.6 B cells to 1.5 x 106 cells/mL in CLICK medium + 5% FBS.
- 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. - 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.
- Carefully aspirate the media on each Ag-coverslip and then add 100 µL of cold 1x PBS to stop the activation.
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Materials
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 |