Overview
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 1x Tyrode buffer and store it at -20 °C. Avoid repeated freezing and thawing.
- 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.
- 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.
- 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).
- 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.
- 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.
- 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.- 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.
Subscription Required. Please recommend JoVE to your librarian.
Materials
Name | Company | Catalog Number | Comments |
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 |