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

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 1x Tyrode buffer and store it at -20 °C. Avoid repeated freezing and thawing.
2. Remove the culture medium from the 24 well plates containing the mucosal mast cell line, rat basophilic leukemia (RBL)-2H3 transfected with Neuropeptide Y fused monomeric RFP ( NPYmRFP) secretory granules reporter gene and wash 3 times with Tyrode buffer. Prepare unstimulated cells by adding 200 μl Tyrode buffer to control wells.
3. Prepare 10 μl/well of 20X concentrated activating reagent [(e.g. 1,000 ng/ml DNP-BSA or DNP-HSA (Ag), 200 µM Ca2+ionophore (e.g. A23187), and a combination of 20 µM Ca²+ ionophore and 1,000 nM 12-O-  tetradecanoylphorbol-13-acetate (TPA)]. If the reagents are stored in DMSO, dilute the reagents into 20x concentration in Tyrode buffer containing 1% DMSO. Incubate at 37 °C for 30 min.
4. Remove the supernatants of each well carefully to a 96-well plate, place on ice, and avoid light. (The supernatants contain the chimeric peptide NPY-mRFP that was released from the cells).
5. Add 200 μl Tyrode buffer containing 0.5% Triton X-100 to each well and incubate at 37 °C for 10 min. (This step is important for the preparation of cell lysates that contain the remaining NPY-mRFP that was not released from the cells). Collect the cell lysates and transfer them to a 96-well plate, place them on ice, and avoid light.
6. Measure the fluorescence of the cell supernatants and cell lysates using a fluorescence plate reader, using a 590-, 20 nm bandwidth excitation filter and 635-, 35 nm bandwidth emission filter.
7. Calculate the percentage of NPY-mRFP released:
   NOTE: The fluorescence reader measures arbitrary fluorescence units (AFU). AFU values depend on the machine, its sensitivity, and the transfection efficiency.
   1. Set the autofluorescence of nontransfected RBL cells as blank. Divide the AFU of each supernatant by the total fluorescence (AFU of the supernatants + AFU of the corresponding lysate) and multiply by 100.