An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells
Published: November 30, 2023
Abstract
Source: Klein, O., et al. Imaging FITC-dextran as a Reporter for Regulated Exocytosis J. Vis. Exp. (2018).
This video demonstrates a technique to visualize exocytosis in mast cells. The cells are incubated with FITC-dextran — a fluorescent reporter — which is taken up by cells inside secretory granules (SGs), where the acidic pH quenches its fluorescence. Upon stimulation of granule exocytosis, the medium's higher pH induces reporter fluorescence inside the fused SGs, which is visualized using a fluorescence microscope.
Protocol
1. Preparations Preparation of RBL (RBL-2H3 mast cell line) culture media 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. Filter the media by using a top-va…
Divulgations
The authors have nothing to disclose.
Materials
| DMEM low glucose | Biological Inductries | 01-050-1A | |
| Fetal bovine serum | Gibco | 12657 | |
| Pen-strep-nystatin solution | Biological Inductries | 03-032-1B | |
| L-Glutamine 200 mM solution | Biological Inductries | 03-020-1A | |
| FITC dextran 150K | Sigma-Aldrich | 46946-500MG-F | |
| Trypsin/EDTA Solution B | Biological Inductries | 03-052-1A | Warm in 37 °C water bath before use |
| Top-vacuum filter of 0.22 µm pore size | Sigma-Aldrich | CLS430769-1EA | |
| Cellulose acetate syringe filter unit, 0.22 µm pore size | Sartorius | 16534K | |
| Chambered coverglass | Thermo scientific | 155411 | |
| Corning tissue-culture treated culture dishes | Sigma-Aldrich | CLS430167 | |
| Bovine serum albumin | Sigma-Aldrich | A4503 | |
| Anti-DNP monoclonal IgE | Sigma-Aldrich | D8406 | |
| DNP-HSA (Ag) | Sigma-Aldrich | A6661 | Avoid direct light exposure |
| Hepes buffer 1M, pH 7.4 | Biological Inductries | 03-025-1B | |
| CaCl2 | MERK | 102382 | |
| Glucose | BDH Laboratories | 284515V | |
| Ammonium chloride | MERK | 1145 | |
| PIPES dipotassium salt | Sigma-Aldrich | 108321-27-3 | |
| Calcium acetate hydrate | Sigma-Aldrich | 114460-21-8 | |
| Magnesium acetate tetrahydrate | Sigma-Aldrich | M5661 | |
| Potassium glutamate (L-Glutamic acid potassium salt monohydrate) | Sigma-Aldrich | G1501 | |
| NaH2PO4 | MERK | 6346 | |
| NaCl | MERK | 106404 | |
| MgCl2 | MERK | 105833 | |
| KCl | MERK | 104936 | |
| Electroporator | BTX | ECM830 | |
| Confocal microscope, Zeiss | Zeiss | LSM 5 Pascal, Axiovert 200M | Used in Figure 3. Equipped with an electronic temperature-controlled |
| airstream incubator | |||
| Confocal microscope, Zeiss | Zeiss | LSM 800, Axio Observer.Z1 /7 | Used in Figure 2. Equipped with a GaAsP detector an electronic temperature-controlled |
| airstream incubator | |||
| Confocal microscope, Leica | Leica | SP5 | Used in Figure 1. Equipped with a leica HyD detector and an top-stage incubator (okolab) |
| RBL-2H3 cells | RBL-2H3 cells were cloned in the lab of Reuben P. Siraganian. See reference 67 |
Citer Cet Article
An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells. J. Vis. Exp. (Pending Publication), e21872, doi: (2023).