Mast Cell Degranulation Assay to Study the Effect of a Target Chemical
Published: July 31, 2023
Abstract
Source: Tsvilovskyy, V., et al. Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays. J. Vis. Exp. (2018).
This video demonstrates a colorimetric assay to determine the degranulation activity of mast cells. The enzyme released by the mast cells hydrolyzes the substrate and produces a colored product. The degranulation activity of the mast cells was measured as the percentage release of enzymes outside the cell normalized with the total enzyme content in the cell.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. PMC Isolation and Cultivation by Intraperitoneal Lavage PMC isolation by intraperitoneal lavage Prior to the cell isolation, prepare the materials listed in Table 1. Use 8 to 14-week-old male mice for PMC isolation. Euthanize a mouse by CO2 in…
Representative Results
Figure 1: Beta-hexosaminidase release colorimetric multi-well measurements performed with PMCs isolated from wild-type mice. (A) Stimulation scheme of 96-well plate pipetting for performing the beta-hexosaminidase assay; results are presented in panel B. "Spontan" corresponds to pipetting of Tyrode's Solution without the addition of any secr…
Divulgazioni
The authors have nothing to disclose.
Materials
| RPMI Medium | Thermo Scientific | 21875034 | |
| DPBS | Sigma-Aldrich | 14190144 | |
| FCS | Thermo Scientific | R92157 | |
| IL-3 | R&D Systems | 403-ML | |
| SCF | Thermo Scientific | PMC2115 | |
| DNP-HSA | Sigma-Aldrich | A6661 | |
| Anti-DNP-Antibody | Sigma-Aldrich | D-8406 | |
| Penstrep | Thermo Scientific | 15140122 | |
| Compound 48/80 | Sigma-Aldrich | C2313 | |
| 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol | Sigma-Aldrich | X100 | |
| 4-Nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside | Sigma-Aldrich | N9376 | |
| CoverWell Imaging Chamber | Sigma-Aldrich | GBL635051 | |
| 96-Well plate, v-shaped bottom | Corning | 3896 | |
| 96-Well plates, flat bottom | Greiner Bio-One | 655180 | |
| Microtiter plate reader with "i-control" software | Tecan | Nano Quant, infinite M200 Pro | |
| Flat bottom plate | Greiner Bio-One | 655180 | |
| Ionomycin | Sigma-Aldrich | I9657 | |
| 50 mL Plastic Centrifuge Tubes | Sarstedt | 62.547.254 | |
| Culture Flasks (25 cm) | Greiner Bio-One | 69160 | |
| Hemocytometer | VWR | 631-0696 | |
| Monochromatic Light Source | Sutter Instruments | Lambda DG-4 | |
| 15 mL Plastic Centrifuge Tubes | Sarstedt | 6,25,54,502 | |
| Bench Centrifuge | Heraeus | Megafuge 1.0R |
Tags
Citazione di questo articolo
Mast Cell Degranulation Assay to Study the Effect of a Target Chemical. J. Vis. Exp. (Pending Publication), e21514, doi: (2023).