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 …

Disclosures

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

Play Video

Cite This Article
Measuring Mast Cell Exocytosis via Neuropeptide Y Monomeric Red Fluorescent Protein Release. J. Vis. Exp. (Pending Publication), e21843, doi: (2023).

View Video