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Measuring Mast Cell Exocytosis via Neuropeptide Y Monomeric Red Fluorescent Protein Release

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.

1. Measuring NPY-mRFP Exocytosis

  1. Preparation of Tyrode buffer:
    1. 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.
    2. 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.
    3. Aliquot the .......

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