Name: isolation of peritoneum-derived mast cells and their functional characterization with ca2+-imaging and degranulation assays
Uploaded: 2018-07-04T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays at JoVE.com

The authors would like to acknowledge Julia Geminn for technical assistance in mast cell preparation and culturing. This work was supported by The Transregional Collaborative Research Centre (TR-SFB)152.

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
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			<td>10 mL syringes</td>
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			<td>27 G needles</td>
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<ol>
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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, ...

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 organs such as the skin, lung, and gut, and are usually found only in small numbers in the blood. MCs derive from hematopoietic precursors, such as MC progenitors, and complete their differentiation and maturation in the microenvironments of almost all vascularized tissues1. T-cell-derived factor interleukin (IL)-3 selectively promotes the viability, proliferation, and differentiation of a pluripotent population of mouse MCs from their hematopoietic progenitors3. Stem cell factor (SCF) is produced by structural cells in the tissues and plays a crucial role in the MC development, survival, migration, and function4. The properties of individual MCs may differ depending on their ability to synthesize and store various proteases or proteoglycans. In mice, the so-called connective tissue-type MCs are distinguished from mucosal MCs according to their anatomic localization, morphology, and content of heparin and proteases5.
In MCs, an increase of free cytosolic Ca2+ ([Ca2+]i) is indispensable for the degranulation and production of eicosanoids, as well as for the synthesis of cytokines and activation of transcription factors in response to antigen and various secretagogues6. A major downstream target of these stimuli is phospholipase C, which hydrolyzes phosphatidylinositol 4,5-bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate. DAG activates protein kinase C and IP3 releases ions of Ca2+ from the endoplasmic reticulum. Depletion of these stores activates Ca2+ influx through plasma membrane Ca2+ channels, leading to store operated calcium entry (SOCE). This process is evoked through the interaction of the Ca2+-sensor, stromal interaction molecule-1 (Stim1), in the endoplasmic reticulum with Orai17 as well as through the activation of transient receptor potential canonical (TRPC) channel proteins (for review see Freichel et al. 20148) in the plasma membrane.
To study the physiological role of these channels, several pharmacological (application of channel blockers) or genetic approaches are typically used. In the latter case, suppression of protein expression is achieved by targeting mRNA (knockdown) or genomic DNA editing with global or tissue-specific9 deletion of an exon coding a pore forming subunit of a channel (knockout). The availability of a blocker with sufficient specificity for these channels is limited. In addition, the knockdown approach requires careful control of its effectivity using, e.g., Western blot analysis, and is hampered by the unavailability of specific antibodies for the targeted channel protein in many cases. Thus, the usage of knockout mouse models is still considered as the gold standard for such analysis. A preferred in vitro model for the investigation of MC functions is cultivation of PMCs that can be isolated ex vivo as a fully mature population (as opposed to differentiating MCs in vitro from, e.g., bone marrow cells)10. As compared to bone marrow derived MCs (BMMCs), which are also commonly used to study MC function in vitro, the stimulation of Fc&#949;RI and beta-hexosaminidase release is increased 8-fold and 100-fold in PMCs, respectively. In this article, we describe a method of isolation and cultivation of murine PMCs, which allows to obtain&#160;after 2 weeks of culturing a high number of pure connective tissue type MCs, sufficient for further analysis.
Despite recent progress in the development and application of genetically encoded calcium indicators, their usage is still limited by difficulties in delivery of genes to target cells, specifically, in highly-specialized cells such as primary cultured MCs. In addition, this group of fluorescent indicators for [Ca2+]i measurements still lacks ratiometric dyes with a high dynamic range. For these reasons, the preferred method for ratiometric [Ca2+]i measurement is still the use of the fluorescent dye &#34;Fura-2&#34;11.
Currently, the most commonly used approach for the evaluation of MC activation and degranulation is the measurement of &#946;-hexosaminidase activity. &#946;-hexosaminidase, an allergic mediator, is one of the MC granule components that is co-released with histamine in constant proportion from MCs12. &#946;-hexosaminidase can be readily and accurately measured through its reaction with a specific substrate, which produces a measurable quantity of a colorigenic product that is easily detectable in a microplate colorimetric assay. In this article, an application of this technique for the analysis of PMCs degranulation in response to a variety of stimuli is reported.
Aggregation of the high-affinity plasmalemmal IgE receptor (Fc&#603;RI) activates in MCs a versatile intracellular signaling pathway, leading to a release of secretory granule content in the surrounding extracellular space. In addition to the specific antigen, many other stimuli can activate MCs to release a diverse array of immunomodulatory mediators, such as complement anaphylatoxins (e.g., C3a and C5a)13, the vasoconstrictor peptide endothelin 1 (ET1)14, as well as numerous cationic substances and drugs provoking pseudoallergic reactions (e.g., icatibant)15 by binding to MRGPRX216. Intracellular signaling pathways involved in MRGPR-induced MCs degranulation are poorly characterized as compared to Fc&#603;RI-mediated intracellular signaling; these pathways only started to be intensively studied during the last few years17 after the receptor identification16. Largely, the plasma membrane ion channels involved in calcium entry followed by MRGPRX2 stimulation remain to be understood. Therefore, the present article also focuses on intracellular calcium signaling and degranulation of MCs stimulated with MRGPR agonists.

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			<td height="40" style="height:40px;width:291px;">RPMI Medium</td>
			<td style="width:273px;">Thermo Scientific</td>
			<td style="width:212px;">21875034</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;">DPBS</td>
			<td>Sigma-Aldrich</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FCS</td>
			<td>Thermo Scientific</td>
			<td>R92157</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IL-3</td>
			<td>R&#38;D Systems</td>
			<td>403-ML</td>
			<td></td>
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			<td height="40" style="height:40px;">SCF</td>
			<td>Thermo Scientific</td>
			<td>PMC2115</td>
			<td></td>
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			<td height="40" style="height:40px;">Concanavalin A</td>
			<td>Sigma-Aldrich</td>
			<td>C2272</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fura-2 AM&#160;</td>
			<td>Thermo Fischer Scientific</td>
			<td>F1221</td>
			<td></td>
		</tr>
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			<td height="40" style="height:40px;">Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNP-HSA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A6661</td>
			<td></td>
		</tr>
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			<td height="40" style="height:40px;">Anti-DNP-Antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D-8406</td>
			<td></td>
		</tr>
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			<td height="40" style="height:40px;">Penstrep</td>
			<td>Thermo Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Compound 48/80&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C2313</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:291px;">4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:212px;">X100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:291px;">4-Nitrophenyl 2-acetamido-2-deoxy-&#946;-D-glucopyranoside</td>
			<td>Sigma-Aldrich</td>
			<td style="width:212px;">N9376</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CoverWell Imaging Chamber</td>
			<td>Sigma-Aldrich</td>
			<td>GBL635051</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">96-Well plate, v-shaped bottom&#160;</td>
			<td>Corning</td>
			<td>3896</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">96-Well plates, flat bottom</td>
			<td>Greiner Bio-One</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microtiter plate reader with &#34;i-control&#34; software&#160;</td>
			<td>Tecan</td>
			<td>Nano Quant, infinite M200 Pro</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flat bottom plate&#160;</td>
			<td style="width:273px;">Greiner Bio-One</td>
			<td style="width:212px;">655180</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:291px;">Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td style="width:212px;">I9657</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">50 mL Plastic Centrifuge Tubes</td>
			<td>Sarstedt</td>
			<td style="width:212px;">62.547.254&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Culture Flasks (25 cm)</td>
			<td style="width:273px;">Greiner Bio-One</td>
			<td>69160</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Cover slips glasses round; &#248; 25 mm; No. 1</td>
			<td>Menzel</td>
			<td>CB00250RA1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Hemocytometer</td>
			<td>VWR</td>
			<td>631-0696</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Inverted Microscope</td>
			<td>Zeiss</td>
			<td>Observer Z1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Objectiv Fluar 40x/1.3 Oil</td>
			<td>Zeiss</td>
			<td style="width:212px;">440260-9900</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">HC Filter Set Fura 2&#160;</td>
			<td>AHF</td>
			<td style="width:212px;">H76-521</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CCD Camera</td>
			<td>Zeiss</td>
			<td>AxioCam MRm&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Monochromatic Light Source</td>
			<td>Sutter Instruments</td>
			<td>Lambda DG-4</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Imaging Acquisition Program</td>
			<td>Zeiss</td>
			<td>AxioVision 4.8.2&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Gravity fed solution application system</td>
			<td>Custom Made</td>
			<td>4-channel</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">15 mL Plastic Centrifuge Tubes</td>
			<td>Sarstedt</td>
			<td>62,554,502</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:291px;">Bench Centrifuge</td>
			<td style="width:273px;">Heraeus</td>
			<td style="width:212px;">Megafuge 1.0R</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
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isolation of peritoneum-derived mast cells and their functional characterization with ca2+-imaging and degranulation assays

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 (Fc&#949;RI), 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 &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-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.

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 &#176;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...

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

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 &#946;-hexosaminidase.

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

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 ...

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