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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