An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells

1. Preparations

1. Preparation of RBL (RBL-2H3 mast cell line) culture media
   1. Mix 500 mL of low glucose Dulbecco's modified Eagle's medium (DMEM) with 56 mL of fetal bovine serum (FBS), 5.5 mL of penicillin-streptomycin-nystatin solution, 5.5 mL of L-Glutamine 200 mM solution. This results in low glucose DMEM supplemented with 10% FBS, 100 µg/mL streptomycin, 100 units/mL penicillin, 12 units/mL nystatin, and 2 mM L-glutamine.
   2. Filter the media by using a top-vacuum filter of 0.22 µm pore size and store at 4 °C.
2. Maintenance of RBL cells.
   1. Grow RBL cells to a maximum confluency of 90% in a 10 cm dish. If the cell culture is healthy, the cells should have a spindle shape with occasional protrusions.
   2. For cell splitting, detach the cells from the dish by aspirating the media and replacing it with 2 mL of trypsin/EDTA solution B. Incubate for 5-10 minutes in a humidified atmosphere of 5% CO₂ at 37 °C.
   3. Once the cells have detached, neutralize the trypsin by adding 2 mL of culture media, using a pipette, and split the cells in a 1:2 – 1:10 ratio.
3. Preparation of 20x Tyrode's buffer
   1. Prepare a stock 20x solution of 54 mM KCl, 20 mM MgCl₂, 2.74 M NaCl, and 8 mM NaH₂PO₄ in double distilled water (DDW). Mix well and store at 4 °C.

2. Culture of RBL Cells for Live Cell Microscopy

1. Preparation of FITC-dextran solution.
   1. Mix 1 mg of fluorescein isothiocyanate (FITC)-dextran powder (150 K) per 1 mL of culture media (see step 1.1.1). For a full chambered coverglass, prepare 3 mL.
   2. Using a cellulose acetate syringe filter unit with 0.22 µm pore size, filter the dissolved FITC-dextran.
   3. Add mouse immunoglobulin E (IgE) to a concentration of 1 µg/mL.
2. Seeding RBL cells for imaging
   1. The day before imaging, aspirate the media from the culture dish and replace it with 2 mL of trypsin/ethylenediaminetetraacetic acid (EDTA) solution B. Incubate for 5 – 10 minutes in a humidified atmosphere of 5% CO₂ at 37 °C. Once the cells have detached, neutralize the trypsin by adding 2 mL of culture media.
   2. Count RBL cells using a hemocytometer and adjust the volume accordingly with culture media to get a cell concentration of 7.5 x 10⁵/mL.
   3. Add 10 µL of the cell suspension to a chambered coverglass pre-filled with fresh FITC-dextran supplemented culture media (this results in the seeding of 7.5 x 10³ cells in a chamber).
   4. Grow RBL cells overnight in a humified atmosphere of 5% CO₂ at 37 °C. The cells should remain at a sub-confluent level in order to make sure that the cells are separated and that it is easy to identify each cell individually under the microscope.
3. Transfection of RBL cells – optional.
   1. For imaging exocytic events in combination with other fluorescently tagged proteins, see transfection protocol for RBL cells in Azouz et al.

3. Live Cell Microscopy of Exocytosis

1. Preparation of solutions:
   1. Prepare a final Tyrode's buffer solution by diluting the stock solution in DDW in a 1:20 dilution and supplement with 20 mM Hepes pH 7, 1.8 mM CaCl₂, 1 mg/mL bovine serum albumin (BSA), and 5.6 mM glucose.
   2. Freshly prepare a 20x secretagogue reagent in Tyrode's buffer [1 µg/mL dinitrophenyl conjugated to human serum albumin (DNP-HAS (Ag)) in our case, for a 50 ng/mL 1x concentration].
   3. Prepare a 400 mM ammonium chloride solution by dissolving the powder in Tyrode's buffer.
2. Preparation of the cells.
   1. Wash the chambered coverglass 3 times by aspirating the media from the chamber and refilling it with 300 µL of Tyrode's buffer, prewarmed to 37 °C. Finally, replenish the chamber with 300 µL of Tyrode's buffer, prewarmed to 37 °C.
   2. Place the chambered coverglass in the microscope's incubator chamber. Make sure that the chamber is stable.
3. Setting up the microscope
   1. To choose a region of interest to track, turn on the fluorescent light source (traditionally a mercury lamp) and choose the appropriate fluorescence filter (choose the filter for green fluorophores for viewing FITC-dextran). Once the region of interest is in focus and is in the middle of the field of view, turn off the light source in order to avoid photo-bleaching and toxicity.
      ​NOTE: Some of the FITC-dextran incorporated in the cells may retain fluorescence. This is because FITC-dextran may also be sorted into non-lysosomal compartments such as endosomes.
   2. Turn on the appropriate lighting for FITC excitation. If using a laser-based microscope, turn on the 488 nm laser. Emission should be gathered around 500 – 550 nm (FITC emission peaks at 510 – 520 nm).
   3. When using a confocal microscope, open the pinhole to the maximum. This will allow the use of lower laser power to avoid bleaching and toxicity and will ensure the capture of exocytosis events from all planes of the cell.
   4. Calibrate the time interval between image acquisitions.
      1. Different cells may vary in their kinetics of regulated exocytosis. For specific imaging of compound exocytosis, we recommend a time interval of at least 5 seconds. However, as a rule of thumb, to allow for fast imaging, make sure that the acquisition time of a single frame is fast.
      2. When using a laser-scanning confocal microscope, set the scan direction to bi-directional, do not allow for averaging, and set the resolution at 512 x 512 (recommended; the latter two provisions will also help to minimize bleaching and toxicity).
4. Imaging of exocytosis.
   1. Image cells for desired durations, depending on the type of cell or secretagogue. In RBL cells triggered with IgE/Ag, most exocytic events occur within 15 – 20 min of activation.
   2. For activation of the cells, add 16 µL from the 20x secretagogue solution to the chamber.
5. Confirming FITC-dextran presence in the cells.
   1. To confirm the presence and localization of FITC-dextran to the SGs, add 16 µL of ammonium chloride solution (be careful not to move the chamber in order not to lose focus) to the chamber gently (this will make a final concentration of 20 mM). This will induce alkalization of the SGs and dequenching of FITC fluorescence, which will occur within seconds.