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

Published: August 31, 2023

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 Preparation of antigen-coated beads 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. <li…

Açıklamalar

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
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

Etiketler

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Bu Makaleden Alıntı Yapın
Organelle Dynamics in B Lymphocytes following Activation with Antigen-Coated Beads. J. Vis. Exp. (Pending Publication), e21610, doi: (2023).

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