This study investigates the exocytosis of neuropeptide Y (NPY) in mast cells using a fusion protein, NPY-mRFP. The methodology involves inducing calcium influx to facilitate the release of NPY-mRFP from secretory granules.
Take transfected mast cells expressing NPY-mRFP, a fusion protein containing neuropeptide Y and monomeric red fluorescent protein, in their cytoplasmic secretory granules.
Remove the media. Pipette calcium-containing buffer with an activating reagent, a calcium ionophore, and incubate.
These ionophores induce calcium influx, increasing the intracellular calcium concentration and mobilizing secretory granules to the plasma membrane.
The calcium binds to calcium-binding proteins on the secretory granule membrane.
Further, the secretory granules fuse with the plasma membrane mediated by membrane-bound proteins, resulting in exocytosis and release of contents, including NPY-mRFP.
Post-incubation, collect and transfer the released NPY-mRFP-containing supernatant to a black multi-well plate.
Add a non-ionic detergent to lyse the cells and release the remaining intracellular NPY-mRFP.
Collect and transfer the cell lysate to the multi-well plate.
Using a fluorescence plate reader, measure the fluorescence of the cell supernatant and lysate to quantify the percentage of NPY-mRFP released and determine the extent of mast cell exocytosis.
To measure NPY-mRFP exocytosis, remove the culture medium from the 24-well plate, and wash three times with Tyrode buffer. Prepare unstimulated cells by adding 200 microliters of Tyrode buffer to the control wells. Then, add the activating reagents diluted in Tyrode buffer before incubating at 37 degrees Celsius for 30 minutes.
Carefully transfer the supernatants of each well to a 96-well plate. Place the plate on ice, and protect from light. The supernatants contain the chimeric peptide NPY-mRFP that was released from the cells.
Next, add 200 microliters of Tyrode buffer containing 0.5% Triton X-100 to each well, and incubate at 37 degrees Celsius for 10 minutes. This step is important for preparation of cell lysates that contain the remaining NPY-mRFP that was not released from the cells. Collect the cell lysates and transfer to a 96-well plate before placing on ice in the absence of light.
Measure the fluorescence of the cell supernatants and cell lysates using a fluorescence plate reader with a 590-nanometer, 20-nanometer bandwidth excitation filter, and 635-nanometer and 35-nanometer bandwidth emission filter.
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