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 ...

These methods can help answer key questions in the field of intracellular signaling on excitable cells, such as which ion channels are critical for immune cell activation and mediator release. The main advantages of these techniques are their relative simplicity and the possibility of simultaneous analysis of large number of cells. Demonstrating Fura-2 calcium imaging in mast cells will be Kathrin Ohlenschlager, a graduate student from my laboratory.To begin this procedure, spray the euthanized mouse with 70%ethanol and fix it on a foam block with its dorsal side down using pins. Using blunt-edge scissors, remove the skin over the abdomen of the mouse and avoid damaging the peritoneal cavity. Next, inject seven milliliters of ice-cold RPMI medium in the peritoneal cavity by inserting a 27 gauge needle carefully in the peritoneum.Do not perforate any organs, and use a spot in the region of the epididymal fat to reduce the risk of an organ perforation. After injection, shake the mouse for one minute to detach the peritoneal cells into the RMPI medium. Do not shake the mouse too strongly to avoid damaging the internal organs and contaminating the peritoneal cavity with the blood.Then, reuse the syringe by equipping it with a new 20 gauge cannula. Shift the inner organs to one side by tilting the foam block and gently tapping it on the bench to make medium aspiration easier from the other side. Subsequently, insert a 20 gauge needle bevel-up to aspirate the fluid from the abdomen gently and slowly to avoid clogging by the inner organs.Collect as much fluid as possible. Then, remove the needle from the syringe and transfer the collected cell suspension in a collection tube on ice. Discard a sample tube if there is visible blood contamination.Centrifuge the tube with clean cell suspension at 300 g for five minutes. Under a sterile hood, aspirate the supernatant. Re-suspend the sample pellets in cold PMC medium and transfer the cell suspension to a 25-square-centimeter cell culture flask.Subsequently, add the growth factors IL-3 and SCF to the final concentrations of 10 nanograms per milliliter and 30 nanograms per milliliter, respectively. Incubate the cells for approximately 48 hours at 37 degrees Celsius until the next procedure. On days 12 through 15, if any experiments with stimulation of plasmalemmal IgE receptors are planned, pretreat the cells overnight with 300 nanograms per milliliter of IgE anti-DNP in standard culture medium.To prepare Fura-2 imaging, dilute Fura-2, AM stock solution in calcium HBSS to a final concentration of five micromolar in order to prepare the loading buffer. Then, mix 500 microliters of the loading buffer with 500 microliters of the PMC cell suspension. Pipette the mixture into the imaging chamber and incubate the cells for 20 to 30 minutes at room temperature in the dark.Set up the gravity-fed application system with different solutions according to the stimulation protocol. Next, mount the application system on the stage of the inverted microscope. Prepare a 40X high-aperture oil immersion objective by placing a small drop of immersion oil on it.Place the imaging chamber with the loaded cells in the application system and secure it to prevent movement during recording. Then, turn on the transmitted light and focus on the cells. Subsequently, start the imaging acquisition program in the Physiological Acquisition mode.Open the setup window and adjust the focus and the optimal acquisition time. Image a reference picture and mark the cells as ROIs. Mark the last ROI in an area where no cells are present and define it as the background.Then, complete the setup and start the measurement with a five-seconds-acquisition rate. Next, add solutions at the proper cycle number according to the design of the experiment. To measure antigen-induced elevation of intracellular calcium concentration, apply the syringe-two solution after cycle 20 and stop recording after cycle 150.To measure compound 48/80-induced elevation of intracellular calcium concentration, apply the syringe-three solution after cycle 20 and stop recording after cycle 150. To measure the elevation of intracellular calcium concentration evoked by SOCE, apply the syringe-four solution after cycle 10, apply the syringe-five solution after cycle 70, apply the syringe-four solution after cycle 120, apply the syringe-one solution after cycle 150, and stop recording after cycle 220. In the acquisition program, consecutively press the buttons Start Cutter, Mark All, Convert All, Physiology Measurements, OK, and then OK again.Save the files as tables and image stacks for offline analysis. To measure beta-hexosaminidase release, transfer 100 microliters of the cell suspension per well to a 96-well V-bottom well plate. Add 25 microliters of either stimulation or control solution to each well.Then, incubate the cells for 45 minutes at 37 degrees Celsius. Afterward, stop the reaction by placing the 96-well plate on ice for five minutes. Then, centrifuge the plate at four degrees Celsius for four minutes at 120 g.Transfer 120 microliters of the supernatant to a flat-bottom 96-well plate and place it on ice. Carefully avoid touching the cell pellets, but completely aspirate the supernatant. Next, add 125 microliters of lysis buffer to the cell pellets.Incubate the cell pellets for five minutes at room temperature and re-suspend them after the incubation by repeated pipetting. Pipette 25 microliters of the four-millimolar pNAG solution in the required wells of a new flat-bottom 96-well plate. Add 25 microliters of each supernatant and 25 microliters of each cell lysate separately to the prepared pNAG solution.Incubate the reaction batches for one hour at 37 degrees Celsius. After the incubation, pipette 150 microliters of stop solution to each well to stop the reaction. Next, analyze the plate sample light absorbance with a micro-plate reader at 405 nanometers with reference 630 nanometers for automatic background subtraction.In the acquisition program, check the box Reference, and in the absorbance program element menu, select 630 nanometers for automatic background subtraction. Beta-hexosaminidase release assay was performed to test the ability of the isolated PMCs that undergo degranulation upon crosslinking of the plasmalemmal IgE high-affinity receptors or stimulation of the Mrg PRB2 receptors. The cells were added to a V-bottom 96-well plate and stimulated according to this scheme for 45 minutes at 37 degrees Celsius.After centrifugation and removal of the supernatant, the pellets were lysed. The beta-hexosaminidase content of individual supernatants and pellet lysates was then analyzed by their reactions with pNAG during a one-hour incubation at 37 degrees Celsius. The amount of the reaction products was detected colorimetrically and the percentage of the released beta-hexosaminidase was calculated.The spontaneous release was very low, whereas the responses to strong degranulation stimuli, such as 10 micromolar ionomycin and compound 48/80 at 50 micrograms per milliliter, were over 40%and 60%respectively. Degranulation responses to DNP stimulation were dose-dependent over the concentration range of 10 to 300 milligrams per milliliter. Together, these data clearly indicate a good functional state of the isolated PMCs.Using this technique, you will obtain highly pure population of murine mature connective tissue mast cells derived from peritoneum within two weeks. While attempting this procedure, it's important to remember to maintain sterile conditions during the cell isolation and culturing to avoid bacterial contamination. Following this procedure, other methods, like electrophysiological patch-clamp ion current measurements can be performed in order to answer additional questions concerning calcium entry pathways in mast cells.After its development, Fura-2 intracellular calcium imaging technique paved the way for researchers in the field of mass cell physiology to explore calcium dependence of mast cell degranulation. After watching this video, you should have a good understanding of how to perform Fura-2 calcium imaging experiments and beta-hexosaminidase degranulation assay with mast cells. Don't forget that working with UV radiation can be hazardous for eyes and skin, and unnecessary exposure should be avoided while performing this procedure.

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