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

JoVE Encyclopedia of Experiments

Immunologie

0 weergaven • 3:49 min • July 8th, 2025

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Take bead-supported lipid bilayers, or BSLBs, silica beads coated with lipid bilayers conjugated with antigenic peptide-MHC complexes, adhesion and co-stimulatory molecules, acting as synthetic antigen-presenting cells.

Incubate with T cells.

TCRs bind to antigenic peptides on BSLBs, which, along with co-stimulatory signals, form an immune synapse, activating T cells.

This binding triggers intracellular signaling cascades, inducing actin rearrangement and forming microclusters at the synapse containing adhesion and signaling molecules.

T cells release trans-synaptic vesicles enriched in TCRs, co-stimulatory molecules, and lytic granule contents into the synapse, binding to BSLBs.

Gradually cool down the co-culture to separate BSLBs from T cells.

Centrifuge and discard the supernatant. Use a protein-containing buffer to block the free binding sites on the surfaces.

Centrifuge and treat BSLBs and T cells with fluorophore-conjugated antibodies that bind to specific signaling and co-stimulatory molecules.

Using flow cytometry, analyze fluorescence signals from BSLBs and T cells to quantify the synaptic output.

Resuspend 500,000 BSLBs per well in 200 microliters of HBS/HSA buffer. Transfer 100 microliters of BSLBs per well to a new U-bottom 96-well plate to make a duplicate, such that the final amount of BSLB per well is 250,000. Spin down the BSLBs at 300 times g for two minutes at room temperature. Discard the supernatant, and then, resuspend the BSLBs using 100 microliters of T cell suspension. Mix gently to prevent the formation of bubbles. Incubate the co-cultures for 90 minutes at 37 degrees Celsius.

Protect the cells from light, and cool down the co-cultures by first incubating them at room temperature for a minimum of 15 minutes. Centrifuge the co-cultures at room temperature for 5 minutes at 500 times g. Discard the supernatant and resuspend the co-cultures in calcium and magnesium ion-free 2% BSA-PBS at room temperature for blocking. Place the cells on ice for 45 minutes, and protect them from light.

Prepare the antibody master mix using ice-cold 0.22-micrometer-filtered 2% BSA and PBS as a staining buffer. This master mix will provide extra blocking. Spin down the co-cultures at 500 times g for 5 minutes and 4 degrees Celsius. Discard the supernatants, and then, use a multichannel pipette to resuspend the cells in the standing master mix containing optimized antibody concentrations.

Include isotype-labeled cells and BSLBs, fluorescent and non-fluorescent BSLBs, and cells and BSLBs stained alone. Mix gently by pipetting up and down half of the volume. Incubate for 30 minutes on ice and protect them from light. Wash the cells and BSLBs twice using ice-cold 2% BSA-PBS Spin them down at 500 times g for 5 minutes at 4 degrees Celsius. After centrifugation, check the co-cultures sedimented on the bottom of the wells. Then, resuspend the washed co-cultures in 100 microliters of PBS and acquire immediately.

09:56

Super-resolutie beeldvorming van de Natural Killer Cell Immunological Synapse op een glas-ondersteunde Planar lipidendubbellaag

Gerelateerde video's

0 Bekeken

07:11

Dissipatief Microgravimetry te bestuderen van de dynamiek van de Binding van de fosfolipide bindend proteïne Annexine A2 aan lipide Solid-ondersteund Bilayers met behulp van een Resonator Quartz

Gerelateerde video's

0 Bekeken

12:18

Assemblage van cellimitiek ondersteunde en gesuspendeerde lipide bilayer modellen voor de studie van moleculaire interacties

Gerelateerde video's

0 Bekeken

12:24

Ondersteunde Planar dubbellagen voor de vorming van studie van immunologische synapsen en Kinapse

Gerelateerde video's

0 Bekeken

11:45

Imaging van HIV-1 Envelope-geïnduceerde Virologische Synapse en signalering op synthetische lipidendubbellagen

Gerelateerde video's

0 Bekeken

16:10

Een TIRF Microscopie Techniek voor Real-time, gelijktijdige beeldvorming van de TCR en de bijbehorende signaaleiwitten

Gerelateerde video's

0 Bekeken

05:29

An In Vitro Technique to Study T Cell Interaction with Supported Planar Lipid Bilayers

Gerelateerde video's

0 Bekeken

19:05

Het meten van TCR-pMHC Binding In Situ Met een FRET-gebaseerde Microscopie Assay

Gerelateerde video's

0 Bekeken

09:25

Een endotheliale Planar Cell Model for Imaging Immunologische Synapse Dynamics

Gerelateerde video's

0 Bekeken

10:34

Ligand Nano-cluster Arrays in een ondersteunde lipide dubbellaag

Gerelateerde video's

0 Bekeken

Laatst bijgewerkt: 15 augustus 2026