Encyclopedia of Experiments: Immunology
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Transcript
Begin with a multi-well plate containing platelet-rich plasma. Platelets express surface mechanoreceptor complexes — responsive to mechanical stimuli.
The mechanoreceptor complex contains the extracellular ligand-binding domain and macro-glycopeptide region linked to the mechanosensory domain.
Introduce anti-mechanoreceptor antibodies, cross-linking with the ligand-binding domains of the receptors on opposing platelets.
Place a droplet of the antibody-treated plasma suspension between the assemblies of the viscometer.
Apply shear stress, simulating mechanical force, pulling the antibody-bound receptors away from the platelets, leading to macro-glycopeptide region dissociation from the mechanosensory domain.
This triggers a conformational change in the mechanosensory domain, activating platelets and resulting in the expression of surface markers.
Transfer the sheared platelet suspension to a new well within the multi-well plate.
Introduce a cocktail of fluorophore-labeled marker-specific molecules interacting with corresponding platelet surface markers and fix the cells with a fixative solution.
In flow cytometry, distinct fluorescence signals correlate with the effects of shear stress on antibody-treated platelets.
