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…

Divulgations

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

Play Video

Citer Cet Article
Mast Cell Degranulation Assay to Study the Effect of a Target Chemical. J. Vis. Exp. (Pending Publication), e21514, doi: (2023).

View Video