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