Measuring Mast Cell Exocytosis via Neuropeptide Y Monomeric Red Fluorescent Protein Release
Published: November 30, 2023
Abstract
Source: Azouz, N. P., et al., Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis. J. Vis. Exp. (2015)
This video demonstrates mast cell exocytosis induced by an activating reagent that includes a calcium ionophore, initiating the release of secretory granules containing the fluorescent reporter protein NPY-mRFP. Subsequently, we quantitatively assess the exocytosis by measuring the released NPY-mRFP using a fluorescence plate reader.
Protocol
1. Measuring NPY-mRFP Exocytosis Preparation of Tyrode buffer: Prepare a solution of 54 mM KCl, 20 mM MgCl2, 2.74 M NaCl, and 8 mM NaH2PO4 in DDW. Mix well and store at 4 °C. This step is for the preparation of 20x Tyrode buffer. Prepare a solution of 20 mM Hepes pH 7, 1.8 mM CaCl2, 1 mg/ml BSA, 5.6 mM glucose, and 1 to 20 dilutions of Tyrode 20x in DDW and mix well. This step is for the preparation of 1x Tyrode buffer. Aliquot the …
Divulgazioni
The authors have nothing to disclose.
Materials
| DMEM | Sigma-Aldrich | D6046-500ML | Warm in 37 °C water bath before use |
| Fetal Bovine Serum | GE health care Life sciences | SH30071.01 | |
| Penicillin-Streptomycin | Life technologies | ||
| Cellulose acetate membrane, pore size 0.22 μm | Sigma-Aldrich | CLS430769-1EA | |
| Corning tissue-culture treated culture dishes | Sigma-Aldrich | CLS430167 | |
| Trypsin/EDTA Solution (TE) | Life technologies | R001100 | Warm in 37 °C water bath before use |
| 24 well, flat bottom | Sigma-Aldrich | CLS3524 | |
| Corning 96-well plates | Sigma-Aldrich | CLS3367 or CLS390 | |
| 96-well plate fluorescence readerInfinite 200 | Tecan | ||
| Triton-X-100 | Sigma-Aldrich | T8787 |
Citazione di questo articolo
Measuring Mast Cell Exocytosis via Neuropeptide Y Monomeric Red Fluorescent Protein Release. J. Vis. Exp. (Pending Publication), e21843, doi: (2023).