B Cell Activation on an Antigen-Coated Coverslip

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…

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