Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Summary

We describe a method for the generation of in vitro derived mast cells, their engraftment into mast cell-deficient mice, and the analysis of the phenotype, numbers and distribution of engrafted mast cells at different anatomical sites. This protocol can be used to assess the functions of mast cells in vivo.

Abstract

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such as skin, airways and gastrointestinal tract. Best known for their detrimental role in IgE-dependent allergic reactions, MCs have also emerged as important players in host defense against venom and invading bacteria and parasites. MC phenotype and function can be influenced by microenvironmental factors that may differ according to anatomic location and/or based on the type or stage of development of immune responses. For this reason, we and others have favored in vivo approaches over in vitro methods to gain insight into MC functions. Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into genetically MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This method, named the ‘mast cell knock-in’ approach, has been extensively used over the past 30 years to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.

Introduction

Mast cells (MCs) are hematopoietic cells that arise from pluripotent bone marrow progenitors^1-3. Following bone marrow egression, MCs progenitors migrate into various tissues where they develop into mature MCs under the influence of local growth factors^1-3. Tissue-resident MCs are strategically located at host-environment interfaces, such as the skin, the airways and the gastrointestinal tract, where they behave as a first line of defense against external insults^3-6. MCs are often sub-classified based on their “baseline” phenotypic characteristics and their anatomic locations. In mice, two types of MCs have been described: ....

Protocol

All animal care and experimentation were conducted in compliance with the guidelines of the National Institutes of Health and with the specific approval of the Institutional Animal Care and Use Committee of Stanford University.

1. Generation and Characterization of Bone Marrow-derived Cultured Mast Cells (BMCMCs).

Note: Donor BMCMCs should be generated from bone marrow cells of the same genetic background as the recipient MC-deficient mice. Male-derived donor BMCMCs are not suitable for engraftment of female mice. Female-derived donor BMCMCs will successfully engraft into both male and female recipients.

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Results

An overview of the ‘mast cell knock-in’ approach is shown in Figure 1, and includes the generation of BMCMCs, the number of cells that should be engrafted i.p., i.d. or i.v. into MC-deficient mice (the number can be varied if indicated based on the experimental design) and the interval between engraftment and experiment depending on the injection site (this interval also can vary, if indicated; e.g., the content of stored mediators in MC cytoplasmic granules increases steadily with time.......

Discussion

Almost 30 years after its initial description³⁸, the ‘mast cell knock-in’ approach continues to provide valuable information about what MCs can do or can’t do in vivo. The functions of MCs were long thought to be limited to their role in allergy. Data generated using the ‘mast cell knock-in’ approach have changed this view, by providing evidence that MCs can, among other functions, play critical roles in host defense against certain pathogens^4,39

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1% Antibiotic-Antimycotic Solution	Corning cellgro	30-004-Cl
3 ml Syringe	Falcon	309656
35 mm x 10 mm Dish	Corning cellgro	430588
5 ml Polystyrene Round Bottom Tube	Falcon	352058
Acetic Acid Glacial	Fisher Scientific	A35-500
Alcian Blue 8GX	Rowley Biochemical Danver	33864-99-2
Allegra 6R Centrifuge	Beckman
Anti-mouse CD16/32 (clone 93) Purified	eBioscience	14-0161-81
2-Mercaptoethanol	Sigma Aldrich	M7522
BD 1 ml TB Syringe	BD Syringe	309659
BD 22 G x 1 (0.7 mm x 25 mm) Needles	BD Precision Glide Needle	205155
BD 25 G 5/8 Needles	BD Syringe	305122
BD 30 G x 1/2 Needles	BD Precision Glide	305106
Blue MAX Jr, 15 ml Polypropylene Conical Tube	Falcon	352097
Chloroform	Fisher Scientific	C298-500
Cytoseal 60 Mounting Medium	Richard-Allan Scientific	8310-4
Cytospin3	Shandon	NA
DakoCytomation pen	Dako	S2002
Dulbecco Modified Eagle Medium (DMEM) 1x	Corning cellgro	15-013-CM
Ethanol	Sigma Aldrich	E 7023-500ml
Fetal Bovine Serum Heat Inactivated	Sigma Aldrich	F4135-500ml
FITC Conjugated IgG2b K Rat Isotype Control	eBioscience	14-4031-82
Fluorescein Isotiocyanate (FITC) Conjugated Anti-mouse KIT (CD117; clone 2B8)	eBioscience	11-1171-82
Formaldehyde	Fisher Scientific	F79-500
Giemsa Stain Modified	Sigma Aldrich	GS-1L
Isothesia	Henry Schein Animal Health	29405
May-Grunwald Stain	Sigma Aldrich	MG-1L
Multiwell 6 well plates	Falcon	35 3046
Olympus BX60 Microscope	Olympus	NA
Paraplast Plus Tissue Embedding Medium	Fisher Brand	23-021-400
PE Conjugated IgG Armenian Hamster Isotype Control	eBioscience	12-4888-81
Phosphate-Buffered-Saline (PBS) 1x	Corning cellgro	21-040-CV
Phycoerythrin (PE) Conjugated Anti-mouse FceRIa (clone MAR-1)	eBioscience	12-5898-82
Propidium Iodide Staining Solution	eBioscience	00-6990-50
Recombinant Mouse IL-3	Peprotech	213-13
Safranin-o Certified	Sigma Aldrich	S8884
Tissue culture flasks T25 25 cm2	Beckton Dickinson	353109
Tissue culture flasks T75 75 cm2	Beckton Dickinson	353110
Toluidine Blue 1% Aqueous	LabChem-Inc	LC26165-2
Recombinant Mouse SCF	Peprotech	250-03