This article describes a method for studying mast cell exocytosis using pH-sensitive fluorescent reporters. The process involves the binding of IgE antibodies to mast cells, followed by antigen exposure that triggers the release of secretory granules.
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.
Add 10 microliters of the cell suspension to a chambered coverglass with fresh FITC-dextran supplemented culture media, and grow the RBL cells overnight. First, prepare a final Tyrode's buffer solution according to the text protocol. Then, prepare a 20x secretagogue reagent in the freshly-made buffer.
Dissolve ammonium chloride powder in Tyrode's buffer to create a 400-millimolar ammonium chloride solution. Next, aspirate the media from the chambered coverglass, and replace it with 300 microliters of pre-warmed Tyrode's buffer. After repeating the washing process two times, replenish the chamber with 300 microliters of Tyrode's buffer.
Next, place the chambered coverglass in the incubator chamber of a microscope, and ensure the chamber is stable. Turn on the fluorescent light source and select the appropriate fluorescence filter. Once the region of interest is in focus in the middle of the field of view, turn off the light source.
Then, turn on the 488-nanometer laser with the emission gathered around 500 to 550 nanometers. Next, calibrate the time interval between image acquisitions. Set the scan direction to "Bi-directional." Do not allow for averaging. Open the pinhole, and set the resolution at 512 by 512.
Next, image the cells for the appropriate duration for the specific cell type or secretagogue. Then, add 16 microliters from the secretagogue solution to the chamber and continue filming. Finally, to confirm the presence and localization of FITC-dextran to the secretory granules, gently add 16 microliters of ammonium chloride solution to the chamber.
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