Encyclopedia of Experiments: Immunology
Se requiere una suscripción a JoVE para ver este contenido. Inicie sesión o comience su prueba gratuita.
Transcript
Begin with a functionalized glass coverslip with polyethylene glycol-anchored gold nanoparticles.
Add DNA hairpin tension probes containing a DNA hairpin hybridized with two oligonucleotides in opposite arms.
The ligand strand includes a fluorophore and biotin, while the anchor strand contains a quencher and a thiol group. In this setup, the quencher suppresses fluorescence.
The DNA probes bind to the gold nanoparticles, anchoring to the glass coverslip.
Introduce streptavidin, interacting with the biotin molecules of the ligand strands.
Add biotinylated ligands that bind to pre-bound streptavidin molecules.
Overlay the coverslip with CD8-positive T cells; their receptors interact with immobilized ligands, forming a receptor-ligand interaction site.
This interaction generates a force that unfolds the DNA hairpin structure, separating the fluorophore from the quencher and promoting fluorescence emission.
Introduce non-fluorescent locking strands that interact with unfolded segments of the probes to preserve the fluorescence signal.
Quantify the fluorescence to assess receptor forces applied by the immune cells.
