An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells
Transcrição
Take a chambered coverglass containing mast cells — immune cells with secretory granules, or SGs, containing inflammatory mediators.
Add IgE antibodies along with a pH-sensitive fluorescent reporter, and incubate.
The antibodies bind to Fc receptors on mast cells. The reporters get internalized via pinocytosis, and the resulting endosomes fuse with SGs.
Remove the media. Add an antigen containing multiple IgE-binding epitopes; place the slide under a fluorescence microscope, and apply the required excitation wavelength.
The acidic pH of SGs quenches reporter fluorescence.
The antigen binds to IgE-receptor complexes on the cell surface, crosslinking them.
Antigen-mediated receptor aggregation triggers SGs to migrate toward the plasma membrane.
SNARE proteins on the plasma and SG membrane mediate membrane fusion and the discharge of SG cargo, termed exocytosis.
The higher pH of the medium causes SG alkalization, inducing the reporters to regain fluorescence.
Incoming SGs fuse with the membrane-fused ones. Alkalization of the second SG mediates fluorescence recovery, confirming compound exocytosis.
