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
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>1</td>
			<td>Ice</td>
		</tr>
		<tr>
			<td>2</td>
			<td>10 mL syringes</td>
		</tr>
		<tr>
			<td>3</td>
			<td>27 G needles</td>
		</tr>
		<tr>
			<td>4</td>
			<td>20 G needles</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Styrofoam block and pins</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Collection tubes (50 mL Plastic Centrifuge Tubes)</td>
		</tr>
		<tr>
			<td>7</td>
			<td>70% ethanol</td>
		</tr>
		<tr>
			<td>8</td>
			<td>RPMI Medium (Pre chilled and kept on ice)</td>
		</tr>
		<tr>
			<td>9</td>
			<td>PMC Medium: RPMI Medium + 20% FCS (Fetal Calf Serum) + 1% Penicillin Streptomycin solution (Pen-Strep)</td>
		</tr>
		<tr>
			<td>10</td>
			<td>Dulbecco&#8217;s phosphate buffered saline - DPBS (without Ca2+ and Mg2+)</td>
		</tr>
		<tr>
			<td>11</td>
			<td>Culture Flasks (25 cm)</td>
		</tr>
		<tr>
			<td>12</td>
			<td>Growth factors stock solutions (IL-3: 1 &#956;g/&#956;L; SCF: 2.5 &#956;g/mL)</td>
		</tr>
		<tr>
			<td>13</td>
			<td>Serological pipettes (10 mL)</td>
		</tr>
		<tr>
			<td>14</td>
			<td>Pipettes tips sterile (20&#8211;200 &#181;L)</td>
		</tr>
		<tr>
			<td>15</td>
			<td>Hemocytometer</td>
		</tr>
		<tr>
			<td>16</td>
			<td>Bench Centrifuge</td>
		</tr>
		<tr>
			<td>17</td>
			<td>Scissors and forceps</td>
		</tr>
		<tr>
			<td>18</td>
			<td>CO2 chamber for mice</td>
		</tr>
		<tr>
			<td>19</td>
			<td>Open sterile hood</td>
		</tr>
		<tr>
			<td>20</td>
			<td>Closed sterile hood</td>
		</tr>
		<tr>
			<td>21</td>
			<td>Cell incubator (37 &#176;C and 5% CO2)</td>
		</tr>
		<tr>
			<td>22</td>
			<td>Transfer pipettes (20&#8211;200 &#181;L)</td>
		</tr>
	</tbody>
</table>
Table 1: Materials for Step 1.
<ol>
	<li>PMC isolation by intraperitoneal lavage
	<ol>
		<li>Prior to the cell isolation, prepare the materials listed in Table 1.</li>
		<li>Use 8 to 14 week-old male mice for PMC isolation. Euthanize a mouse by CO2 inhalation, and confirm the death by loss of reflexes. Spray the mouse with 70% ethanol and fix it on a foam block using pins (dorsal side down). Remove the ventral skin of the mouse using blunt edge scissors. Avoid damaging the peritoneal cavity. 
		NOTE: We use typically this protocol for mice with a C57BL/6N strain genetic background.</li>
		<li>Inject 7 mL of the ice-cold RPMI medium and 5 ml of air in the peritoneal cavity using a 10 mL syringe equipped with a 27 G needle. Push the needle carefully in the peritoneum and do not perforate any organs. Use a spot in the region of the epididymal fat to reduce the risk of an organ perforation.</li>
		<li>After injection, shake the mouse for 1 min to detach peritoneal cells into the RPMI medium. Do not shake the mouse too strongly, to avoid damaging the internal organs and contaminating the peritoneal cavity with the blood.</li>
		<li>Reuse the 10-mL syringe by equipping it with a new 20 G 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. Insert a 20 G needle, bevel up, and aspirate the fluid from the abdomen gently and slowly (~0.5 mL/s) to avoid clogging by the inner organs. Collect as much fluid as possible (typically 5&#8211;6 mL).</li>
		<li>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.</li>
		<li>Centrifuge the tubes with the cell suspension at ~300 x g for 5 min. Under a sterile hood aspirate the supernatant. For each mouse, combine the sample pellets in 4 mL of cold (4&#8211;10 &#176;C) PMC Medium and transfer the cell suspension to a 25 cm2 culture flask. Add the growth factors IL-3 and SCF to the final concentrations 10 ng/mL and 30 ng/mL, respectively. Place the flask containing the cells in an incubator (37 &#176;C and 5% CO2) and incubate for ~48 h until the next procedure.</li>
	</ol>
	</li>
	<li>PMC culture
	<ol>
		<li>Day 2 (~48 h after isolation): Medium change
		<ol>
			<li>To remove the supernatant and non-adherent cells (erythrocytes and dead cells), aspirate the medium from the flask by placing a Pasteur pipette tip at the edge of the flask and holding the flask almost horizontally. Add 4 mL of pre-warmed PMC Medium per mouse. Add the growth factors IL-3 (10 ng/mL) and SCF (30 ng/mL). Place the flask containing the cells in an incubator (37 &#176;C and 5% CO2).</li>
		</ol>
		</li>
		<li>Day 9: Cell splitting
		<ol>
			<li>Transfer the cell suspension from the cell culture flask into a 50 mL plastic centrifuge tube. Wash the flask three times with 10 mL pre-warmed (37 &#176;C) Dulbecco&#39;s phosphate-buffered saline (DPBS), collecting the cell suspension with the detached cells in the same 50 mL plastic centrifuge tube. Count the cell concentration by a hemocytometer, and calculate the total number of the cells.</li>
			<li>Centrifuge the cell suspension at ~300 x g for 5 min and aspirate the supernatant. Recover the pellet in pre-warmed PMC Medium (37 &#176;C) to obtain the cell concentration 1 x 106 cells/mL. Transfer the cell suspension to a new 25 cm2 culture flask. Discard the old flask with fibroblasts adhering to the bottom.</li>
			<li>Add the growth factors IL-3 (10 ng/mL) and SCF (30 ng/mL), and place the flask containing the cells in an incubator (37 &#176;C and 5% CO2).</li>
		</ol>
		</li>
		<li>Days 12&#8211;15: Cell measurements
		<ol>
			<li>If any experiments with stimulation of Fc&#603;RI receptors are planned, pretreat the cells overnight with 300 ng/mL of IgE anti-DNP in standard culture medium. Proceed with step 2 or step 3.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Fluorometric Intracellular Free Calcium Concentration Measurement in PMCs
<ol>
	<li>Prior to the measurements, prepare the materials listed in Table 2. Also, prepare an Imaging Setup consisting of: Inverted Fluorescence Microscope; Objective: 40X/1.3 Oil; Fura 2 Filter Set; CCD Camera; Excitation Light Source; Imaging Acquisition Program; Gravity fed solution application system.</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>1</td>
			<td>PMC (12&#8211;15 days old) 1 x 106 cells/mL</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Concanavalin A</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Fura-2 AM</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Pluronic F-127 20% solution</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Cover slips glasses round; &#248; 25 mm; No. 1</td>
		</tr>
		<tr>
			<td>6</td>
			<td>DNP-HSA (dinitrophenol-human serum albumin) stock solution</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Anti-DNP-Antibody (IgE) stock solution</td>
		</tr>
		<tr>
			<td>8</td>
			<td>Flat bottom plate (12 wells)</td>
		</tr>
		<tr>
			<td>9</td>
			<td>Compound 48/80 stock solution</td>
		</tr>
		<tr>
			<td>10</td>
			<td>Cover Well Imaging Chambers</td>
		</tr>
		<tr>
			<td>11</td>
			<td>Hemocytometer</td>
		</tr>
		<tr>
			<td>12</td>
			<td>Transfer pipettes (20&#8211;200 &#181;L)</td>
		</tr>
	</tbody>
</table>
Table 2: Materials for Step 2.
<ol start="2">
	<li>Fura-2 imaging preparation
	<ol>
		<li>Perform a cell count using a hemocytometer and adjust the cell concentration to 1 x 106 cells/mL. Split the PMCs colonies by gently pipetting (3&#8211;5 times) the cell suspension with a 1 mL pipette tip.</li>
		<li>Prepare a Ca2+-containing HEPES buffered salt solution (Ca2+-HBSS) composed of 135 mM NaCl, 6 mM KCl, 2 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES, and 12 mM glucose. Adjust the pH to 7.4 with 3 M NaOH.</li>
		<li>Prepare concanavalin A coated slides by pipetting 1 drop of concanavalin A solution (0.1 mg/mL in H2O) on each 25 mm round cover glass under a sterile hood. Leave the drops on the cover slips for at least 1 min, then remove most of the solution with a pipette and let the cover slips air-dry for 45 min. Carefully press the concanavalin A treated cover slips on the rubber imaging chamber (see Table of Materials) until they attach to obtain microscopy imaging chambers. 
		NOTE: Poly-L-Lysine coated slides can be used as an alternative.</li>
		<li>Dissolve the fluorescent Ca2+ dye Fura-2 AM (1 mM) in dimethyl sulfoxide (DMSO). Store this solution protected from light.</li>
		<li>Dilute the Fura-2 AM stock solution in Ca2+-HBSS to a final concentration of 5 &#181;M in order to prepare the &#34;loading buffer&#34;. Supplement with 5 &#181;L/mL of 20% pluronic F-127 in DMSO.</li>
		<li>Mix 500 &#181;L of the &#34;loading buffer&#34; with 500 &#181;L of the PMC cell suspension. Pipette the mixture into the imaging chamber and incubate the cells for 20&#8211;30 min at room temperature in the dark.</li>
		<li>Set up the gravity fed application system with different solutions according to the stimulation protocol. Supplement syringe 1 with 40 mL of Ca2+-HBSS solution, syringe 2 with 40 mL of Ca2+-HBSS solution containing 100 ng/mL DNP, syringe 3 with 40 mL of Ca2+-HBSS solution containing 50 &#181;g/mL Compound 48/80, syringe 4 with 40 mL of nominally Ca2+-free-HBSS solution, and syringe 5 with 40 mL of nominally Ca2+-free-HBSS solution containing 2 &#181;M Thapsigargin.</li>
		<li>Prepare a 40X high-aperture oil immersion objective by placing a small drop of immersion oil on it.</li>
		<li>Mount the application system on the stage of the inverted microscope. Put the imaging chamber with the loaded cells in the application system and secure it to prevent movement during recording. Turn on the transmitted light and focus the cells.</li>
		<li>Rinse the cells three times with Ca2+-HBSS buffer using the gravity-fed application system.</li>
		<li>Incubate the cells in Ca2+-HBSS for 5 min for Fura-2 AM de-esterification.</li>
		<li>Rinse the cells one more time before starting the imaging, to remove any potentially leaked fluorescent dye.</li>
	</ol>
	</li>
	<li>Cell imaging
	<ol>
		<li>Start the imaging acquisition program in the physiological acquisition mode. Open the setup window, and adjust the focus and the optimal acquisition time (which should be the same for excitation with 340 nm and 380 nm and typically ranges between 50&#8211;150 ms). 
		NOTE: Minimize the exposure of the Fura-2 loaded cell to any light to avoid photobleaching of the dye.</li>
		<li>Image a reference picture, and mark the cells as regions of interest (ROI). Mark the last ROI in an area where no cells are present and define it as the background.</li>
		<li>Complete the setup and start the measurement with a 5 s acquisition rate. 
		NOTE: The cells will be alternately illuminated with the excitation light of 340 nm and 380 nm, and the emitted fluorescence (&#62;510 nm) will be collected using the CCD camera. The images of fluorescence signals F340 nm and F380 nm as well as the ratio (F340 nm/F380 nm) will be displayed in three windows. The charts of time course of individual ROIs fluorescence intensity mean values will be also continuously displayed.</li>
		<li>Add solutions at the proper cycle number according the design of the experiment:
		<ol>
			<li>To measure antigen-induced elevation of [Ca2+]i, as illustrated in Figure 2A, 2B, apply the syringe 2 solution after cycle 20, and stop recording after cycle 150.</li>
			<li>To measure Compound 48/80-induced elevation of [Ca2+]i,, as illustrated in Figure 2C, apply the syringe 3 solution after cycle 20, and stop recording after cycle 150.</li>
			<li>To measure the elevation of [Ca2+]i evoked by SOCE, as illustrated in Figure 2D, apply the syringe 4 solution after cycle 10, apply the syringe 5 solution after cycle 70, apply the syringe 4 solution after cycle 120, apply the syringe 1 solution after cycle 150, and stop recording after cycle 220.</li>
		</ol>
		</li>
		<li>After finishing the measurement, convert the results into a ratio table containing the measured intensities for both excitation wavelengths with and without background correction, as well as the ratio values with and without background correction for every ROI and every measurement time point. In the acquisition program, consecutively press the buttons: &#34;start cutter&#34;, &#34;mark all&#34;, &#34;convert all&#34;, &#34;physiology measurements&#34;, &#34;Ok, &#34;Ok&#34;. Save the files as tables and image stacks for off-line analysis.</li>
	</ol>
	</li>
</ol>
3. Beta-hexosaminidase Release Measurement in PMCs
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>1</td>
			<td>PMC (12&#8211;15 days old) 1 x 106 cells/mL</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Anti-DNP-Antibody (IgE) stock solution</td>
		</tr>
		<tr>
			<td>3</td>
			<td>DNP-HSA stock solution</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Compound 48/80 stock solution</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Ionomycin</td>
		</tr>
		<tr>
			<td>6</td>
			<td>96-Well plate, v-shaped bottom</td>
		</tr>
		<tr>
			<td>7</td>
			<td>96-Well plates, flat bottom</td>
		</tr>
		<tr>
			<td>8</td>
			<td>Plastic centrifuge tubes (15 mL)</td>
		</tr>
		<tr>
			<td>9</td>
			<td>Serological pipettes</td>
		</tr>
		<tr>
			<td>10</td>
			<td>Cell incubator (37 &#176;C and 5% CO2)</td>
		</tr>
		<tr>
			<td>11</td>
			<td>Bench Centrifuge</td>
		</tr>
		<tr>
			<td>12</td>
			<td>Microtiter plate reader for optical density measurements</td>
		</tr>
		<tr>
			<td>13</td>
			<td>Multichannel pipette (20&#8211;200 &#956;L)</td>
		</tr>
		<tr>
			<td>14</td>
			<td>Hemocytometer</td>
		</tr>
	</tbody>
</table>
Table 3: Materials for Step 3.
<ol>
	<li>Prior to the measurements, prepare the materials listed in Table 3. 
	NOTE: &#946;-hexosaminidase released from MCs after their stimulation hydrolyzes p-nitrophenyl-acetyl-D-glucosamine (pNAG) into p-nitrophenol and N-acetyl-D-glucosamine. The amount of &#946;-hexosaminidase in the sample is proportional to the amount of p-nitrophenol that will be formed. In a high pH environment of &#34;Stop Solution&#34;, p-nitrophenol exists as fully deprotonated p-nitrophenolat and can be detected by its light absorbance at 405&#160;nm.</li>
	<li>Prepare Tyrode&#39;s solution containing 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5.6 mM glucose, and BSA 0.1%. Adjust the pH to 7.4 with 3 M NaOH.</li>
	<li>Prepare the lysis solution by adding a 1% volume of Triton X-100 to the Tyrode&#39;s solution.</li>
	<li>Prepare the stop solution composed of 200 mM glycine and adjust the pH to 10.7 with NaOH.</li>
	<li>Prepare the pNAG solution (4-Nitrophenyl 2-acetamido-2-deoxy-&#946;-D-glucopyranoside) solution by dissolving pNAG in 0.4 M citric acid to a final concentration of 4 mM.</li>
	<li>Calculate the amount of the cells needed for the assay (perform the assay in duplicate). Use 200,000 PMCs per well. Use 2 wells as negative controls (Tyrode&#39;s solution without any secretagogues such as DNP or compound 48/80) and 2 wells as positive controls (Tyrode&#39;s solution with 10 &#181;M Ionomycin) for every genotype.</li>
	<li>Transfer the cells into a 15-mL plastic centrifuge tube, adjust the volume to 15 mL with Tyrode&#39;s solution, and centrifuge at 200 x g for 4 min. Remove the supernatant with a Pasteur pipette and resuspend the cells in Tyrode&#39;s solution to 2 x 106 cells/mL.</li>
	<li>Transfer 100 &#181;L of the cell suspension per well to a 96-well V-bottom well plate. Add 25 &#181;L of either stimulation or control solution to each well (according to the pipetting scheme illustrated in Figure 3A). Incubate the cells for 45 min at 37 &#176;C and 5% CO2.</li>
	<li>Stop the reaction by placing the 96-well plate on ice for 5 min. Then, centrifuge the plate at 4 &#176;C for 4 min at 120 x g. Transfer 120 &#181;L of the supernatant using a multichannel pipette to a flat-bottom 96-well plate and place it on ice. Carefully avoid touching the cell pellets, but completely aspirate the supernatant.</li>
	<li>Add 125 &#181;L of lysis buffer to the cell pellets. Incubate the cell pellets for 5 min at room temperature and resuspend them after the incubation by repeated pipetting (approximately 5 times).</li>
	<li>Pipette 25 &#181;L of the pNAG solution (4 mM) in the required wells of a new flat-bottom 96-well plate. Add 25 &#181;L from each supernatant to the prepared pNAG solution as well as 25 &#181;L of each cell lysate.</li>
	<li>Incubate the reaction batches for 1 h at 37 &#176;C. After the incubation, pipette 150 &#181;L of stop solution to each well to stop the reaction.</li>
	<li>Analyze the plate with a microplate reader using a dual-wavelength setting at 405 nm with reference 630 nm for automatic background subtraction. In the acquisition program, tick the box &#34;Reference&#34; and in the &#34;Absorbance&#34; program element menu, select &#34;630 nm&#34; from the drop-down list. If the optical density of the samples is too high, prepare an appropriate dilution of the probe before remeasuring.</li>
	<li>Calculate the percentage of &#946;-hexosaminidase release according to the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/57222/57222eq1.jpg" /> 
	where Release [%] is the percent of specific beta-hexosaminidase release, A[supernatant] is the background corrected absorption of the supernatant, and A[lysate] is the background corrected absorption of the corresponding cell lysate.</li>
</ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> J Biol Chem. 260 (6), 3440-3450 (1985).</li><li>Schwartz, L. B., Austen, K. F., Wasserman, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+release+of+beta-hexosaminidase+and+beta-glucuronidase+from+purified+rat+serosal+mast+cells.">Immunologic release of beta-hexosaminidase and beta-glucuronidase from purified rat serosal mast cells.</a> J Immunol. 123 (4), 1445-1450 (1979).</li><li>Schafer, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell+anaphylatoxin+receptor+expression+can+enhance+IgE-dependent+skin+inflammation+in+mice.">Mast cell anaphylatoxin receptor expression can enhance IgE-dependent skin inflammation in mice.</a> J Allergy Clin Immunol. 131 (2), 541-549 (2013).</li><li>Maurer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells+promote+homeostasis+by+limiting+endothelin-1-induced+toxicity.">Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.</a> Nature. 432 (7016), 512-516 (2004).</li><li>Maurer, M., Church, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+skin+responses+induced+by+icatibant+injection+are+mast+cell+mediated+and+attenuated+by+H(1)-antihistamines.">Inflammatory skin responses induced by icatibant injection are mast cell mediated and attenuated by H(1)-antihistamines.</a> Exp Dermatol. 21 (1), 154-155 (2012).</li><li>McNeil, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+mast-cell-specific+receptor+crucial+for+pseudo-allergic+drug+reactions.">Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug reactions.</a> Nature. 519 (7542), 237-241 (2015).</li><li>Gaudenzio, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+activation+signals+induce+distinct+mast+cell+degranulation+strategies.">Different activation signals induce distinct mast cell degranulation strategies.</a> J Clin Invest. 126 (10), 3981-3998 (2016).</li><li>Solis-Lopez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+TRPV+channel+activation+by+stimulation+of+FCepsilonRI+and+MRGPR+receptors+in+mouse+peritoneal+mast+cells.">Analysis of TRPV channel activation by stimulation of FCepsilonRI and MRGPR receptors in mouse peritoneal mast cells.</a> PLoS One. 12 (2), 0171366 (2017).</li><li>Enerback, L., Svensson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+peritoneal+mast+cells+by+centrifugation+on+density+gradients+of+Percoll.">Isolation of rat peritoneal mast cells by centrifugation on density gradients of Percoll.</a> J Immunol Methods. 39 (1-2), 135-145 (1980).</li><li>Bird, G. S., DeHaven, W. I., Smyth, J. T., Putney, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+store-operated+calcium+entry.">Methods for studying store-operated calcium entry.</a> Methods. 46 (3), 204-212 (2008).</li><li>Aronson, N. N., Kuranda, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+degradation+of+Asn-linked+glycoproteins.">Lysosomal degradation of Asn-linked glycoproteins.</a> FASEB J. 3 (14), 2615-2622 (1989).</li><li>Moon, T. C., Befus, A. D., Kulka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell+mediators:+their+differential+release+and+the+secretory+pathways+involved.">Mast cell mediators: their differential release and the secretory pathways involved.</a> Front Immunol. 5, 569 (2014).</li></ol>

The authors have nothing to disclose.

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.

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

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...

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

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 ...

isolation-peritoneum-derived-mast-cells-their-functional

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

12.2K Views.  Ruprecht-Karls Heidelberg University. 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.

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

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Developmental Biology

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

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

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 &#8216;mast cell knock-in&#8217; 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.

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

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.

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

Medicine

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

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.

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.

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the ...

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Biology

Mast Cell Degranulation Assay to Study the Effect of a Target Chemical

Source: Tsvilovskyy, V., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/57222">Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays.</a>&#160;J. Vis. Exp. (2018).
This video demonstrates a colorimetric assay to determine the degranulation activity of mast cells. The enzyme released by the mast cells hydrolyzes the substrate and produces a colored product. The degranulation activity of the mast cells was measured as the percentage release of enzymes outside the cell normalized with the total enzyme content in the cell.

Test degranulacji komórek tucznych w celu zbadania wpływu docelowej substancji chemicznej

Source: Tsvilovskyy, V., et al. Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Assays and Functional Profiling

A Technique to Isolate Primary Mast Cells from a Murine Peritoneal Cavity

Source: Tsvilovskyy, V., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/57222">Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays.</a> J. Vis. Exp. (2018).
This video demonstrates a peritoneal lavage method for isolating mature connective tissue-type mast cells from the peritoneal cavity of adult mice. Upon isolating a mixture of primary immune cells from the peritoneal cavity, selective proliferation of mast cells is performed under appropriate culture conditions.

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

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Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

Test degranulacji komórek tucznych w celu zbadania wpływu docelowej substancji chemicznej

Technika izolowania pierwotnych komórek tucznych z mysiej jamy otrzewnowej