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. 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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.

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			<td height="40" style="height:40px;width:291px;">RPMI Medium</td>
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			<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>
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			<td>Thermo Scientific</td>
			<td>PMC2115</td>
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			<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>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-DNP-Antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D-8406</td>
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			<td height="40" style="height:40px;">Penstrep</td>
			<td>Thermo Scientific</td>
			<td>15140122</td>
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		</tr>
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			<td height="40" style="height:40px;">Compound 48/80&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C2313</td>
			<td></td>
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		<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>
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		<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>
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		<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>
</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

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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 Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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 ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...