Mast Cell Degranulation Assay to Study the Effect of a Target Chemical

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

1. PMC isolation by intraperitoneal lavage
   1. Prior to the cell isolation, prepare the materials listed in Table 1.
   2. Use 8 to 14-week-old male mice for PMC isolation. Euthanize a mouse by CO₂ inhalation, and confirm the death by loss of reflexes. Spray the mouse with 70% ethanol and fix it on a foam block using pins (dorsal side down). Remove the ventr....

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 ass.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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