Utilizing Bead-Supported Lipid Bilayers to Investigate the Synaptic Output from T Cells
Published: February 29, 2024
Abstract
Source: Céspedes, P. F., et al. Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses. J. Vis. Exp. (2022)
This video illustrates the reconstitution of synthetic antigen-presenting cells through Bead-Supported Lipid Bilayers and their application in assessing the synaptic output generated by activated T cells.
Protocol
1. Protein density calibrations on BSLBs (Bead-supported lipid bilayers) Before taking the 5.00 ±0.05 µm diameter non-functionalized silica beads, mix the stock solution well and resuspend any big clumps of beads sedimented on the bottom of the flasks. NOTE: Silica beads tend to sediment quickly, which might lead to counting errors. Mix vigorously by pipetting up and down half of the maximum volume of a P1000 micropipette. Dilute 1 µL of bead s…
Representative Results
Figure 1: Absolute quantification of proteins on the surface of APCs. (A) Example of quantitative flow cytometry measurements of ICAM-1 on the surface of tonsillar B cells (Foll. Bc) and helper T cells (TFH). (i-vii) Gating strategy for analyzing single CXCR5+ Bc and TFH isolated from human palatine tonsils. Shown is the sequential gating strategy for identifying…
Divulgazioni
The authors have nothing to disclose.
Materials
| 96 Well Cell Cultture Plate U-bottom with Lid, Tissue culture treated, nonpyrogenic | Costar® | 3799 | For FCM staining and co-culture of BSLB and cells. |
| 96 Well Cell Cultture Plate V-bottom with Lid, Tissue culture treated, nonpyrogenic. | Costar® | 3894 | For FCM staining of cells or beads in suspension. |
| Alexa Fluor 488 NHS Ester (Succinimidyl Ester) | Thermo Fisher Scientific, Invitrogen™ | A20000 | |
| Alexa Fluor 647 NHS Ester (Succinimidyl Ester) | Thermo Fisher Scientific, Invitrogen™ | A37573 and A20006 | |
| Multiwell 6 well Tissue culture treated with vacuum gas plasma | Falcon | 353046 | For culturing and expanding purified CD4+ and CD8+ T cells. |
| Casein from bovine milk, suitable for substrate for protein kinase (after dephosphorylation), purified powder | |||
| Dynabeads Human T-Activator CD3/ CD28 | ThermoFisher Scientific, Gibco | 11132D | |
| DynaMag-2 | ThermoFisher Scientific, Invitrogen™ | 12321D | For the removal of Dynabeads Human T-Activator CD3/CD28 in volumes less than 2 mL |
| DynaMag™-15 | ThermoFisher Scientific, Invitrogen™ | 12301D | For the removal of Dynabeads™ Human T-Activator CD3/CD28 in volumes less than 15 mL |
| Fetal Bovine Serum Qualified, One Sho | ThermoFisher Scientific, Gibco | A3160801 | Needs heat inactivation for 30 min at 56 oC |
| anti-human CD154 (CD40L), clone 24-31 | BioLegend | 310815 and 310818 | Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively |
| Armenian Hamster IgG Alexa Fluor 647 Isotype control, clone HTK888 | BioLegend | 400902 | Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester) |
| BD Cytometer Setup and Tracking beads | Becton Dickinson & Company (BD) | 641319 | Performance track of instruments before quantitative FCM |
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Citazione di questo articolo
Utilizing Bead-Supported Lipid Bilayers to Investigate the Synaptic Output from T Cells. J. Vis. Exp. (Pending Publication), e21992, doi: (2024).