An Ex vivo Mast Cell Degranulation Assay using Crude Peritoneal Exudate Cells and Natural Antigen Stimulation

Summary

We have established an ex vivo mast cell degranulation assay carried out by incubating crude peritoneal exudate cells isolated from the mice, treated with a pharmacological agent of interest and administered anti-dinitrophenol (DNP) IgE beforehand, with DNP on a carrier protein.

Abstract

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the effect of a pharmacological agent of interest on mast cells in vivo. As the anaphylactic symptoms are primarily attributed to exocytosis of the granules from mast cells, it is conceived that the agent to cause amelioration of the symptoms has a mast cell stabilizing activity. Despite the fact, it is prudent to confirm the activity by directly demonstrating the decline in the functional activity of mast cells following its treatment. In vitro degranulation assays using an immortalized mast cell line or cultured primary mast cells are routinely employed to that end. The results from the in vitro and in vivo assays may not always be akin to each other; however, as treatment conditions (e.g., treatment dose, time, surrounding environments) for the in vitro assays are often distinct from those for the in vivo assay such as PSA. In pursuit of an in vitro (or ex vivo) assay to reflect more closely the effect of a pharmacological agent on mast cells in vivo, we devised the ex vivo mast cell degranulation assay in which crude peritoneal exudate cells (PECs) isolated from the mice, treated with the agent and administered anti-dinitrophenol (DNP) IgE, were incubated directly with DNP on a carrier protein. It turned out that the assay was not only useful in validating the mast cell stabilizing activity of a pharmacological agent indicated by the in vivo assay but also practical and highly reproducible.

Introduction

Mast cells play a central role in allergy^1,2. When IgE located on the surface of mast cells via interaction with the high-affinity receptor for IgE (FcεRI) encounters a cognate allergen, a signaling cascade is elicited to prompt the release of the granules. As a result, a variety of allergy effector molecules, including monoamines (e.g., histamine, serotonin), cytokines (e.g., TNF-α), and proteolytic enzymes (e.g., tryptase, chymase), are released to cause a series of immunological, neurological and vasomuscular reactions^3,4.

Protocol

All animal experiments were performed in accordance with the guideline provided by the IACUC (Institutional Animal Care and Use Committee) of Chungnam National University (Animal Protocol Number: CNU-00996).

1. Quantifying mast cell-specific molecules in the lysate of crude PECs

1. Isolate the cells from the mouse peritoneal cavity¹².
   1. Anesthetize a mouse (8 weeks old, male, BALB/C) with isoflurane. Euthanize via cervical dislocation.
   2. Place the mouse on a foam block. Wipe the abdomen with 70% ethanol.
   3. Cut the ventral skin longitudinally with blunt edge scissors. Peel off the ....

Results

Determining the optimal number of PECs for ex vivo mast cell degranulation assay

Mast cells (c-kit⁺·IgE⁺ double positive cells)¹⁵ represent only about 2% of PECs (Figure 1A). Estimating the maximum levels of mast cell-specific molecules to be detected in the culture supernatants on the assumption that 100% of the granules were released by mast cells in PECs, we measured the amounts of β-hexosaminidase<.......

Discussion

The finding that mast cell degranulation assay can be carried out with a relatively small number of crude mouse PECs is significant. Even though PECs must be an excellent source of primary mouse mast cells, it is demanding to purify mast cells in PECs. Although a density gradient media such as Percoll²⁵ has been successfully used for purification of mast cells from rat PECs, its use for purification of mouse peritoneal mast cells has been limited presumably for the difference in the densities of r.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL syringe		1757589701
1.5 mL micro tube	Hisol	MT-15003
10 mL syringe		1757593161
15 mL conical tube	Thermo Fisher scientific	14-959-53A
20xPBS	Tech & Innovation	BPB-9121-500mL
4-nitrophenyl-N-acetyl-β-D-glucosaminide	SIGMA	N9376
5 mL polystyrene round-bottom tube	Life sciences	352003
50 mL conical tube	Thermo Fisher scientific	14-959-49A
Aluminium Fiol	BioFact	TS1-3330
Anti-mouse CD117(c-kit)	Biolegend	135129	keep at 2-8°C
Anti-mouse IgE mAbs	Thermo Fisher scientific	11-5992-81	keep at 2-8°C
Antiti-DNP-IgE	SIGMA	D8406-.2MG	keep at -20°C
Centrifuge	HANIL	396150
D-(+)-gluouse	SIGMA	G8270
Dexamethasone	SIGMA	D2915-100MG
DNP-BSA	Invitrogen	2079360	keep at -20°C
EDTA	Biofact	PB131-500
Fetal Bovine serum	Thermo Fisher scientific	11455035
Gelatin	SIGMA	G1890
Glycine	JUNSEI	27185-0350
hemocytometer	ZEISS	176045
HEPES	Thermo Fisher scientific	15630130
Histamine ELISA kit	Abcam	GK3275957-4	keep at 2-8°C
Hotplate stirrer	Lab teach	zso-9001
Isoflurance	Troikaa	I29159
ketotifen fumarate salt	SIGMA	K2628
MCPT-1 ELISA kit	Thermo Fisher scientific	88-7503-22	keep at 2-8°C
Mouse Fc block	BD Biosciences	553141	keep at 2-8°C
Propidium iodiole	SIGMA	81845	keep at 2-8°C
RBC lysis buffer	Biolegend	420301
Round-bottom 96 well	SPL-life sciences	30096
Single use syringe filter	Startoriusag	16555
Streptavidin microbeads	MilteryiBiotec	130-048-101	keep at 2-8°C
Triton X-100	JUNSEIchemical	49415-1601
TWEEN 20	SIGMA	9005-64-5
Water bath	CHANGSHINSCIENCE	190107