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

Abstract

Source: Ibañez-Vega, J., et al., Studying Organelle Dynamics in B Cells During Immune Synapse Formation. J. Vis. Exp. (2019)

In this video, we describe a method to study the organelle dynamics in B lymphocytes following their activation with antigen-coated microbeads.

Protocol

NOTE: The following steps were performed using IIA1.6 B cells.

1. B cell activation with antigen-coated beads

1. Preparation of antigen-coated beads
   1. To activate B cells, use NH₂-beads covalently coated with antigen (Ag-coated beads), which are prepared using 50 µL (~20 x 10⁶ beads) of 3 µm NH₂-beads with activating (BCR-ligand+) or non-activating (BCR-ligand-) antigens.
   2. For IIA1.6 B cells use anti-IgG-F(ab')₂ fragment as BCR-ligand+ and anti-IgM-F(ab')₂ or bovine serum albumin (BSA) as BCR-ligan....

Materials

List of materials used in this article

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
100% methanol	Fisher Scientific	A412-4
10-mm diameter cover glasses thickness No. 1 circular	Marienfield-Superior	111500
Amino-Dynabeads	ThermoFisher	14307D
Anti-pericentrin	Abcam	ab4448	1:200 dilution recomended but should be optimized
Anti-rab6	Abcam	ab95954	1:200 dilution recomended but should be optimized
Anti-sec61	Abcam	ab15575	1:200 dilution recomended but should be optimized
BSA	Winkler	BM-0150
Polybead Amino Microspheres 3.00μm	Polyscience	17145-5
Poly-L-Lysine	Sigma	P8920	Dilute with sterile water
RPMI-1640	Biological Industries	01-104-1A
Triton X-100	Merck	9036-19-5
Tube 50 ml	Corning	353043