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…
Offenlegungen
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 |
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Diesen Artikel zitieren
Organelle Dynamics in B Lymphocytes following Activation with Antigen-Coated Beads. J. Vis. Exp. (Pending Publication), e21610, doi: (2023).