Utilizing Bead-Supported Lipid Bilayers to Investigate the Synaptic Output from T Cells

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…

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