This method can help answer key questions in the research of regulated exocytosis, by allowing researchers to distinguish between the different modes of exocytosis in the living cell. The main advantage of this technique is that it is simple and requires minimal perturbation to the cells. Though this method can provide insight into mast cell exocytosis, it can also be applied to other secretory cells such as neutrophils and eosinophils.
To begin, prepare a stock solution of 20x Tyrode's buffer according to the text protocol. Next, mix three milligrams of FITC-dextran powder with three milliliters of culture media. Filter the dissolved FITC-dextan with a cellulose acetate syringe filter unit.
Then, add mouse IgE to a concentration of one microgram per milliliter. One day prior to imaging, aspirate media from a culture dish and replace it with two milliliters of Trypsin EDTA Solution B.Then, incubate the dish for five to 10 minutes. Once the cells have detached, add two milliliters of culture media to the dish to neutralize the Trypsin.
Then, use a hemocytometer to count the RBL cells. Adjust the volume of the suspension with culture media to achieve a concentration of 750, 000 cells per milliliter. Next, 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 insure 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 bidirectional, 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. In this protocol, FITC-dextran was used as a reporter for live cell imaging of regulated exocytosis.
Imaging FITC-dextran release allows capturing of the sequential nature of compound exocytosis. After the addition of ammonium chloride to the media, FITC-dextran becomes visible and is co-localized with the mRFP-tagged neuropeptide Y, as secretory granular reporter in RBL cells. While attempting this procedure, it is important to minimize toxicity to the cells while filming by minimizing the exposure of the cells to the laser.
This can be achieved through using the low laser power and short acquisition time. Since compound exocytotic events are longer lived in comparison to other regulated exocytic events, we can categorically capture compound exocytosis by setting a long time interval between acquisitions.
