Measuring Mast Cell Exocytosis via Neuropeptide Y Monomeric Red Fluorescent Protein Release
Transcription
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.
