SR and LDU would like to acknowledge Alberta Innovates Strategic Research Project, NRC, and NSERC-Discovery grant for this project.

1. Design and development of peptide library
<ol>
	<li>To identify the ligands of the mast cell MRGPRX2 receptor based on a known ligand i.e., PAMP-1213, follow the steps below.
	<ol>
		<li>Generate N-truncated peptide library by truncating the N-terminal amino acid residues of the ligand, in succession, by solid-phase peptide synthesis (SPPS).</li>
		<li>Generate C-truncated peptide library by truncating the C-terminal amino acid residues of the known ligand, in succession, by SPPS.</li>
		<li>Based on the results of 1.1.1 and 1.1.2, generate an N+C-truncated peptide library using SPPS by truncating the desired residues from the N and C-terminal, respectively.</li>
		<li>Use solid-phase peptide synthesis to synthesize the peptides13.</li>
		<li>Modify the N-terminal to acetyl (Ac) group and C-terminal to amide group.</li>
		<li>Characterize the peptide for its purity using high-pressure liquid chromatography (HPLC) and for the mass using a mass spectrophotometer.</li>
	</ol>
	</li>
	<li>To study the significance of specific amino acids within the parent PAMP-12 molecule, follow the steps below.
	<ol>
		<li>Generate an alanine scanning peptide library by replacing the respective amino acid residues in the peptide molecule with alanine, one at a time using SPPS. Modify the N-terminal to acetyl (Ac) group and C-terminal to amide group.</li>
		<li>Characterize the peptide for its purity using high-pressure liquid chromatography (HPLC) and for the mass using a mass spectrophotometer. 
		NOTE: Ensure that the synthesized peptides are of high purity. Characterize the peptides using mass spectroscopy and HPLC.</li>
	</ol>
	</li>
</ol>
2.&#160;&#160;In vitro cell culture
<ol>
	<li>Culture HEK-X2 and HEK-WT cells by following the steps below.
	<ol>
		<li>Prepare culture medium by supplementing high glucose DMEM with 10% fetal bovine serum (FBS), 2 mM L-Glutamine, 100 U/mL of penicillin and 100 &#181;g/mL of streptomycin.</li>
		<li>Passage the cells in tissue culture (TC) treated T-75 culture flasks and grow in an incubator at 37 &#176;C containing 5% CO2, till they are 75-80% confluent.</li>
		<li>Once 75% confluent, wash the cells and add 2-3 mL of trypsin for 2 - 3 min. Incubate in a 37 &#176;C, 5% CO2 incubator to detach the cells.</li>
		<li>Once the cells have detached, collect the cells in trypsin. Add 6-9 mL of fresh medium.</li>
		<li>Centrifuge the cells at 1620 x g for 3-5 min.</li>
		<li>After centrifugation, discard the supernatant to collect the pellet. Resuspend the cells in a fresh culture medium. Dilute the cells as per the desired concentration. 
		NOTE: HEK cells are fast-growing cells and hence optimize the cell medium supplements. HEK cells are adherent cells; passage them in TC-treated culture flasks to support adhesion.</li>
	</ol>
	</li>
	<li>Prepare a 96 well assay plate for the experiment.
	<ol>
		<li>Add 200 &#181;L of the cell suspension in each well, with a concentration of 200,000 cells/mL, to seed 40,000 cells/well.</li>
		<li>Grow the cells for 24 h in a 37 &#176;C, 5% CO2 incubator. 
		NOTE: Optimize the cell density per well based on the plate size and the cell strain type. Conduct the experiment in triplicates in a black TC-treated 96 well plate with flat transparent bottom.</li>
	</ol>
	</li>
</ol>
3. Fura-2 AM calcium assay
<ol>
	<li>Prepare dye by following the steps below.
	<ol>
		<li>Use Fura-2 AM dye for the experiment.</li>
		<li>Prepare HEPES-Tyrode&#39;s buffer (HTB) buffer containing 25 mM HEPES buffer, 120 mM NaCl, 5 mM KCl, 1 mg/mL glucose, 1 mg/mL bovine serum albumin (BSA) and freshly added 1.8 mM CaCl2 in autoclave sterilized water.</li>
		<li>Add 50 &#181;L of DMSO in 50 &#181;g Fura-2 AM vial to prepare 1 mM stock solution of Fura-2 AM dye. Add 1 &#181;L of 1 mM Fura-2 AM dye per mL of fresh medium to prepare the dye loading medium having 1 &#181;M dye concentration.</li>
		<li>Remove the 96 well plate from the incubator and discard the medium. Replace the medium with a fresh dye loading medium. Add 200 &#181;L of dye loading medium in each well. Incubate the cells for 30-40 min in a 37 &#176;C, 5% CO2 incubator.</li>
		<li>After 40 min of incubation, remove the medium. Wash the cells with the HTB buffer. Add 100 &#181;L of HTB buffer for fluorescence reading. Take the plate for fluorescence reading. 
		NOTE: Optimize the concentration of dye in the dye loading medium. Dye leakage and photobleaching are possible concerns associated with the dye. Add CaCl2 fresh into the HTB buffer to avoid precipitation.</li>
	</ol>
	</li>
</ol>
4. Cell activation and fluorescent reading
NOTE: Fluorescence plate reader with an automated pipetting system allows for the automatic transfer of compounds from a compound source to the assay plate without taking the plate out of the plate reader.
<ol>
	<li>While the cells are being incubated, set the Plate Reader.
	<ol>
		<li>Set the Temperature to 37 &#176;C.</li>
		<li>In Settings, select Flex.</li>
		<li>Set the Read Mode to Fluorescence and Bottom read.</li>
		<li>In Wavelengths, set the number of Wavelengths to 2. Set the Excitation to 340 nm and 380 nm. Set the Emission to 510 nm.</li>
		<li>Leave the Sensitivity to Default.</li>
		<li>In Timing, set the Interval to 3.9 s. Set Run Time to 94 s to get 25 reads.</li>
		<li>Next, select the Assay Plate Type.</li>
		<li>Next, select the Wells to Read.</li>
		<li>In Compound Transfer, set Transfers to 1 and Initial Volume to 100 &#181;L. Set the Pipette Height to 100 &#181;L, Volume to 50 &#181;L, and Time Point to 36 s, to add the compound at the 10th reading.</li>
		<li>Next, select the Compound Source plate type.</li>
		<li>Leave Triturate to Not Used.</li>
		<li>Select the tips in the Pipette Tips Layout.</li>
		<li>For Compound and Tip Columns, ensure that the compounds to be transferred are in column 1 of the compound plate. Set the Tip Column to 1 and Compound Column to 1.</li>
		<li>Leave the Autocalibrate as ON.</li>
		<li>Click OK.</li>
	</ol>
	</li>
	<li>When the temperature has reached to 37 &#176;C, load the plates into the Plate Reader.
	<ol>
		<li>Press the Reading Chamber to put the Assay Plate into the fluorescence Plate Reader.</li>
		<li>Press the Source to put the Compound Plate. Prepare the Compound Plate by adding 200 &#181;L of respective peptides, ionomycin and ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)-Triton X-100 solutions.</li>
		<li>Press the Tip Rack to put the Tip Box. Use a black tip to avoid tip autofluorescence.</li>
		<li>Once the plates are loaded, review the settings of the software and press Read.</li>
	</ol>
	</li>
</ol>
5. Data analysis
<ol>
	<li>Determine the calcium concentration from the fluorescence ratio by the Grynkiewicz equation - 
	<img alt="Equation 1" src="/files/ftp_upload/62448/62448eq01.jpg" /> 
	where, Kd is the dissociation constant of Fura-2 AM, R is the emission ratio after excitation at 340 nm and 380 nm (F340/F380) for respective peptides, Rmax is the maximum fluorescence ratio observed by the addition of 50 &#181;L of 30 &#181;M ionomycin, Rmin is the minimum fluorescence observed by the addition of 50 &#181;L of 100 mM EGTA/2.5%Triton X-100, and F380min and F380max are the absolute fluorescence intensity of Fura-2 AM in calcium free and bound state, respectively. 
	NOTE: The machine dispenses the liquid with some force. Do not set the peptide dispensing height too close to the bottom of the plate; it may detach the cells. Use black pipette tips to avoid autofluorescence. Read the maximum fluorescence and the minimum fluorescence for each plate for each experiment.</li>
</ol>

<ol>
	<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>Subramanian, H., Gupta, K., Ali, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+Mas-related+G+protein-coupled+receptor+X2+on+mast+cell-mediated+host+defense,+pseudoallergic+drug+reactions,+and+chronic+inflammatory+diseases.">Roles of Mas-related G protein-coupled receptor X2 on mast cell-mediated host defense, pseudoallergic drug reactions, and chronic inflammatory diseases.</a> Journal of Allergy and Clinical Immunology. 138 (3), 700-710 (2016).</li><li>R&#229;dinger, M., Jensen, B. M., Kuehn, H. S., Kirshenbaum, A., Gilfillan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation,+isolation,+and+maintenance+of+human+mast+cells+and+mast+cell+lines+derived+from+peripheral+blood+or+cord+blood.">Generation, isolation, and maintenance of human mast cells and mast cell lines derived from peripheral blood or cord blood.</a> Current protocols in immunology. , (2010).</li><li>Thomas, P., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEK293+cell+line:+A+vehicle+for+the+expression+of+recombinant+proteins.">HEK293 cell line: A vehicle for the expression of recombinant proteins.</a> Journal of Pharmacological and Toxicological Methods. 51 (3), 187-200 (2005).</li><li>Subedi, G. P., Johnson, R. W., Moniz, H. A., Moremen, K. W., Barb, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+yield+expression+of+recombinant+human+proteins+with+the+transient+transfection+of+HEK293+cells+in+suspension.">High yield expression of recombinant human proteins with the transient transfection of HEK293 cells in suspension.</a> Journal of Visualized Experiments. (106), e53568 (2015).</li><li>Occhiuto, C. J., 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=Store-operated+calcium+entry+via+STIM1+contributes+to+MRGPRX2+induced+mast+cell+functions.">Store-operated calcium entry via STIM1 contributes to MRGPRX2 induced mast cell functions.</a> Frontiers in Immunology. 10, 3143 (2020).</li><li>Baba, Y., 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=Essential+function+for+the+calcium+sensor+STIM1+in+mast+cell+activation+and+anaphylactic+responses.">Essential function for the calcium sensor STIM1 in mast cell activation and anaphylactic responses.</a> Nature Immunology. 9 (1), 81-88 (2008).</li><li>Hoth, M., Penner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+intracellular+calcium+stores+activates+a+calcium+current+in+mast+cells.">Depletion of intracellular calcium stores activates a calcium current in mast cells.</a> Nature. 355 (6358), 353-356 (1992).</li><li>Assinger, A., Volf, I., Schmid, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+rapid+method+to+quantify+intraplatelet+calcium+dynamics+by+ratiometric+flow+cytometry.">A novel, rapid method to quantify intraplatelet calcium dynamics by ratiometric flow cytometry.</a> PLoS One. 10 (4), 0122527 (2015).</li><li>Roe, M. W., Lemasters, J. J., Herman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Fura-2+for+measurements+of+cytosolic+free+calcium.">Assessment of Fura-2 for measurements of cytosolic free calcium.</a> Cell Calcium. 11 (2-3), 63-73 (1990).</li><li>Malgaroli, A., Milani, D., Meldolesi, J., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fura-2+measurement+of+cytosolic+free+Ca2++in+monolayers+and+suspensions+of+various+types+of+animal+cells.">Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells.</a> The Journal of Cell Biology. 105 (5), 2145-2155 (1987).</li><li>Grynkiewicz, G., Poenie, M., Tsienb, R. 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> Journal of Biological Chemistry. 260 (6), 3440-3450 (1985).</li><li>Lu, L., Parmar, M. B., Kulka, M., Kwan, P., Unsworth, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+peptide+nanoscaffold+that+activates+human+mast+cells.">Self-assembling peptide nanoscaffold that activates human mast cells.</a> ACS Applied Materials and Interfaces. 10 (7), 6107-6117 (2018).</li><li>Lansu, K., 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=In+silico+design+of+novel+probes+for+the+atypical+opioid+receptor+MRGPRX2.">In silico design of novel probes for the atypical opioid receptor MRGPRX2.</a> Nature Chemical Biology. 13 (5), 529-536 (2017).</li><li>Navin&#233;s-Ferrer, 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=MRGPRX2-mediated+mast+cell+response+to+drugs+used+in+perioperative+procedures+and+anaesthesia.">MRGPRX2-mediated mast cell response to drugs used in perioperative procedures and anaesthesia.</a> Scientific Reports. 8 (1), 11628 (2018).</li><li>Johnson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+imaging+of+store-operated+calcium+(Ca+2+)+entry+(SOCE)+in+HEK293+cells+using+Fura-2.">Calcium imaging of store-operated calcium (Ca 2+) entry (SOCE) in HEK293 cells using Fura-2.</a> Calcium Signalling. , 163-172 (2019).</li><li>Tinning, P. W., Franssen, A. J. P. M., Hridi, S. U., Bushell, T. J., McConnell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+340/380+nm+light-emitting+diode+illuminator+for+Fura-2+AM+ratiometric+Ca2++imaging+of+live+cells+with+better+than+5+nM+precision.">A 340/380 nm light-emitting diode illuminator for Fura-2 AM ratiometric Ca2+ imaging of live cells with better than 5 nM precision.</a> Journal of Microscopy. 269 (3), 212-220 (2018).</li></ol>

Authors declare no competing interests.

Calcium signaling is central to mast cell degranulation and has been widely used in the study of receptor-ligand interactions, ligand identification, and drug discovery14. MRGPRX2 is a recently discovered mast cell receptor that has been found to play a key role in many inflammatory diseases like itch, asthma, and atopic dermatitis, among others2. Furthermore, several approved drugs have been shown to elicit an inflammatory response through the MRGPRX2 receptor<sup class='x...

Mast cells are an integral part of the immune system and play a crucial role in both innate and adaptive immune responses. Mast cells are primarily activated either by the binding of an antigen to the immunoglobulin E (IgE) - Fc&#949;RI receptor complex, or by the recently discovered mas related G-protein receptor-X2 (MRGPRX2)1. MRGPRX2 activation has been linked to several immune and inflammatory diseases, and hence, it is important to understand the binding mechanism of the receptor to its ligands2. To do so, a library of small peptide molecules was developed and screened against MRGPRX2 receptors that were overexpressed in HEK cells. In the study, the peptide library was constructed using the simple and versatile techniques of alanine scanning and amino acid truncation. Alanine scanning involves replacing specific amino acids with an alanine residue. Alanine being small and neutral, strips the peptide of the specific properties conferred by the replaced residue and consecutively highlights the significance of the respective physiochemical properties of the amino acid in receptor interactions. On the contrary, in amino acid truncation, peptide sequences are designed such that it lacks one or more amino acid residues from the N-terminal, C terminal, or both. This set of peptides was used to identify the amino acid sequences crucial to MRGPRX2 binding.
Experience with human mast cells lines (LAD-2) has shown that these cells are difficult to culture and maintain in vitro: a doubling time of two weeks, expensive medium supplements, and direct attention required during passaging3. These attributes make the cells unsuitable for large scale screening of potential ligands. Herein, stably transfected HEK cells expressing MRGPRX2 receptor (HEK-X2) were used to screen the peptide library1. HEK-293 cells are widely used and studied for the heterologous expression of surface receptors due to their high transfection efficiency, faster doubling rate, and the need for non-expensive medium supplements to be cultured in laboratory4. The protocol to transfect HEK-293 cell line has been demonstrated and is well established5. HEK-293 cells stably expressing MRGPRX2 receptor (passage 13-19) were activated with the peptides generated through N-truncation, C-truncation, N+C-truncation, and alanine scanning1. Wild type HEK cells (HEK-WT) (passage 16-21) were used as control. Intracellular calcium release upon activation was monitored to study the MRGPRX2 based activation.
Cell activation by MRGPRX2 is followed by a cytosolic calcium mobilization. This regulated intracellular calcium release in mast cells is regulated by the store operated calcium entry (SOCE), coordinated by the stromal interaction molecule 1 (STIM1); and is central to the immune response cascade6,7. Various methods have been used to detect intracellular calcium concentration, including patch-clamps and fluorescent dyes8. Of all the techniques available, fluorometric calcium dyes in conjugation with various detection techniques are being widely used9. Two types of fluorometric dyes that have gained interests are namely, single wavelength dyes like Fluo-4, and dual wavelength ratiometric dyes like Indo -1 and Fura-2. The advantage that dual wavelength ratiometric dyes bring over single wavelength dyes is that the ratiometric dyes correct for experimental errors like dye loading, photo bleaching, and focusing10,11.
Fura-2 acetoxymethyl ester (Fura-2 AM) is a cell permeating, green-fluorescent dye whose excitation shifts to a lower wavelength upon calcium-binding. Experimentally, Fura-2 is excited at 340 and 380 nm, while the emission is recorded at 510 nm. Upon calcium binding, the fluorescent intensity at 340 nm increases while that of 380 nm decreases, as shown in Figure 1. Data is represented as a ratio of fluorescence intensity after excitation at 340 nm (F340) to that of intensity after excitation at 380 nm (F380) i.e., F340/F380. The F340/F380 ratio is proportional to intracellular calcium, the value of which can be calculated by the Grynkiewicz equation12. Since the fluorescence signal is obtained from the excitation of the dye at two wavelengths (340 nm and 380 nm), the ratio of the fluorescence signals corrects for experimental factors like dye loading, dye leakage, photobleaching, and cell densities.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>793939</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96 Well</td>
			<td>Sigma Aldrich</td>
			<td>CLS3603</td>
			<td></td>
		</tr>
		<tr>
			<td>Black Polystyrene Microplate</td>
			<td>Sigma Aldrich</td>
			<td>CLS3603</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fischer</td>
			<td>11995065</td>
			<td>High Glucose</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Thermo Fischer</td>
			<td>D12345</td>
			<td>Sterile, biological grade</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fischer</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexstation 3</td>
			<td>Molecular devices</td>
			<td>FV06060</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2 AM</td>
			<td>Thermo Fischer</td>
			<td>F1221</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>D8270</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Thermo Fischer</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma Aldrich</td>
			<td>I9657</td>
			<td></td>
		</tr>
		<tr>
			<td>L Glutamine</td>
			<td>Thermo Fischer</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Thermo Fischer</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptides</td>
			<td>RS Syntehsis</td>
			<td>Custom</td>
			<td>&#8805;95% pure; 
			N terminal - acetyl group 
			C terminal - amide group</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>12636</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>DOW Chemical</td>
			<td>166704</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Table 1 contains the peptide sequences generated through terminal amino acid truncation and alanine scanning. As shown in Table 1, peptide sequence RKKWNKWALSR lacks N-terminal phenylalanine (F) with respect to its parent PAMP-12 and hence is a representative peptide in N-truncated library. Similarly, in FRKKWNKWALS, PAMP-12 C-terminal serine has been removed, representing a C-truncated peptide library derived from PAMP-12. In N+C-truncated peptide library, amino acid from both N and C-t...

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Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

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 ...

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Immunology and Infection

2.4K Views.  University of Alberta. 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. 

Video: Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm's tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.

Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...