Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Summary

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca²⁺ concentration and a degranulation assay based on colorimetric quantification of the released β-hexosaminidase.

Abstract

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (FcεRI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca²⁺ level ([Ca²⁺]_i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca²⁺ signaling in MCs. Fluorescence-based monitoring of [Ca²⁺]_i is a reliable and commonly used approach to study Ca²⁺ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a β-hexosaminidase release assay. The amount of β-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. β-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca²⁺-imaging and degranulation assays.

Introduction

MCs play a prominent role during innate and acquired immune responses. Specifically, MCs participate in the killing of pathogens, such as bacteria and parasites, and also degrade potentially toxic endogenous peptides or components of venoms (for review see Galli et al. 2008¹). The physiological role of MCs in innate and adaptive immunity is the subject of heated debate. Therefore, the numerous data discrepancies in the studies performed with different MC-deficient mouse models require a systematic re-evaluation of immunological functions of MCs beyond allergy². Mature MCs are mostly localized in tissues and orga....

Protocol

All animal procedures were performed according to the German legislation guidelines for care and use of laboratory animals (officially approved by the Karlsruhe regional council).

1. PMC Isolation and Cultivation by Intraperitoneal Lavage

....

1	Ice
2	10 mL syringes
3	27 G needles
4	20 G needles
5	Styrofoam block and pins
6	Collection tubes (50 mL Plastic Centrifuge Tubes)

Results

PMCs were isolated from 3 wild type mice (C57BL/6N) by intraperitoneal lavage and further cultured in RPMI medium supplemented with 20% FCS and 1% Pen-Strep in the presence of growth factors (IL-3 at 10 ng/mL and SCF at 30 ng/mL) under sterile humidified conditions at 37 °C and 5% CO₂. The medium was changed on days 2 and 9 after the cell isolation. The cell morphology was analyzed using transmission light DIC imaging (see Figure 1A, B

Discussion

MC models can be successfully used to study MC degranulation, chemotaxis, adhesion, as well as to elucidate intracellular signaling transduction pathways involved in MC activation. Some researchers use human MCs models, such as immortalized lines (LAD2 and HMC1) or human MCs derived from cord blood progenitor cells (CD133+) and peripheral blood progenitor cells (CD34+). Others prefer rodent MC models, such as immortalized (rat basophilic leukemia RBL-2H3 MC line) or primary cultured (mouse bone-marrow-derived MCs BMMCs, .......

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
Concanavalin A	Sigma-Aldrich	C2272
Fura-2 AM	Thermo Fischer Scientific	F1221
Pluronic F-127	Sigma-Aldrich	P2443
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
Cover slips glasses round; ø 25 mm; No. 1	Menzel	CB00250RA1
Hemocytometer	VWR	631-0696
Inverted Microscope	Zeiss	Observer Z1
Objectiv Fluar 40x/1.3 Oil	Zeiss	440260-9900
HC Filter Set Fura 2	AHF	H76-521
CCD Camera	Zeiss	AxioCam MRm
Monochromatic Light Source	Sutter Instruments	Lambda DG-4
Imaging Acquisition Program	Zeiss	AxioVision 4.8.2
Gravity fed solution application system	Custom Made	4-channel
15 mL Plastic Centrifuge Tubes	Sarstedt	62,554,502
Bench Centrifuge	Heraeus	Megafuge 1.0R