We thank Irene Hernandez, Trudy Holyst, Dr. Hartmut Weiler (Versiti Blood Research Institute), and Dr. Leggy Arnold (University of Wisconsin-Milwaukee) for providing space and indirect support of this project, and Dr. John McCorvy (Medical College of Wisconsin) for pertinent advice. We thank the National Heart, Lung, and Blood Institute (R15HL127636), the U.S. Dept. of Defense (W81XWH22101), and the National Science Foundation (2223225) for grant support.

All media exchanges/additions made in steps 1 and 2 of the following protocol are performed in a sterile hood. Unless otherwise noted, all plasticware used in the sterile hood must be purchased sterilized and sealed or sterilized appropriately via autoclave.
1. Initiation of EA.hy926 cell line
<ol>
	<li>Acquire EA.hy926 cells.</li>
	<li>Store vial(s) of cells in the vapor phase of a liquid nitrogen tank.</li>
	<li>Prepare DMEM complete medium by first warming DMEM, FBS, and pen/strep (see Table of Materials for specific details) in a 37 &#176;C water bath for 15-30 min. Add 50 mL of FBS and 1 mL of pen/strep to 500 mL of DMEM. Gently mix the solution by inversion. 
	NOTE: Mixing too vigorously causes excessive amounts of bubbles.</li>
	<li>Remove one vial of cells from liquid nitrogen and warm in a 37 &#176;C water bath for 5 min.</li>
	<li>Sterilize the vial of cells and bottle of DMEM complete medium by spraying with alcohol, then move to the sterile hood.</li>
	<li>Use a 1,000 &#181;L pipette to carefully transfer the cells to a 15 mL sterile centrifuge tube. Add 1 mL of DMEM to the vial to rinse and add to the centrifuge tube.</li>
	<li>Make a pellet of cells using a centrifuge (150 &#215; g, 5 min, 22 &#176;C). Remove the medium from the centrifuge tube by aspiration; be careful not to disturb the cell pellet.</li>
	<li>Add 3 mL of DMEM complete medium to the centrifuge tube and gently break apart the pellet by mixing the cell suspension using a 1,000 &#181;L pipette. 
	NOTE: Causing bubbles by mixing too vigorously should be avoided as this could activate or damage the cells.</li>
	<li>Mix 10 &#181;L of the cell suspension with 10 &#181;L of trypan blue in a microcentrifuge tube using a pipette with either 20 &#181;L or 200 &#181;L tips. Add 10 &#181;L of the cell suspension/trypan blue mixture to a hemocytometer and count the cells. 
	NOTE: Using 0.1-10 &#181;L pipette tips can shear the cells. Tips of this size should not be used while handling the cell suspension.</li>
	<li>Add DMEM complete medium to the cell suspension to make a density of 66,000 cells/mL using either a 10 or 25 mL serological pipette. Pipette 15&#160;mL of the cell suspension into T-75 culture flask(s). Ensure equal dispersion of the cell suspension by moving the flask north to south and east to west.</li>
	<li>Incubate the cells until they reach confluency (typically after 4-6 days), as estimated by examination under a microscope. At this point, use the cells in the experiment (beginning with section 2) or seed them into new flasks for expansion to prepare frozen stocks (at a recommended seeding density of 1,000,000 cells in 15 mL of complete medium per T-75 flask). 
	NOTE: Cells can be grown in the culture flasks for as long as 3 weeks without a noticeable difference in shape, health, or performance in the assay. Complete medium should be exchanged every 3-4 days.</li>
</ol>
2. Addition of EA.hy926 cells to 96-well plates
NOTE: Cells from one confluent T-75 culture flask can be used to prepare two or three assay plates or optionally expanded into as many as 15 fresh T-75 flasks. At least five assay plates can be screened using the protocol in sections 4 and 5 in a normal workday. The following instructions describe preparing and testing one assay plate, but additional plates can be prepared by repeating steps 2.2, 2.7, 2.12, and 3.2 to prepare the desired number of assay plates. Most commonly, four assay plates are prepared per day to measure concentration-responses with up to 16 compounds, which requires two T-75 flasks with confluent cells.
<ol>
	<li>Warm DMEM complete medium in a 37 &#176;C water bath for 15-30 min. Sterilize the bottle of DMEM complete medium by spraying it with alcohol, then move it to the sterile hood.</li>
	<li>Add 100 &#181;L of a sterilized 0.4% gelatin solution to all wells of a black-walled, 96-well, clear-bottom plate with a lid. Incubate the plate with the gelatin solution in an incubator (37 &#176;C, 5% CO2) for 30 min.</li>
	<li>Confirm that cells in a T-75 culture flask are 80-100% confluent using an inverted microscope. Remove DMEM complete medium from the T-75 flask via aspiration.</li>
	<li>Wash the cells in the flask with 10 mL PBS and gently move the flask North to South and East to West. Remove the PBS from the flask via aspiration.</li>
	<li>Add 5 mL of cell dissociation solution to the flask and incubate the flask&#160;in an incubator (37 &#176;C, 5% CO2) for ~12 min. Tap the flask after 6 min to help facilitate dissociation. 
	NOTE: Incubating cells for longer than 15 min with the referenced cell dissociation agent causes cells to begin to clump together, which makes obtaining an accurate cell count more difficult and may interfere with the assay.</li>
	<li>Add 5 mL of the prewarmed DMEM complete medium to the flask and gently mix into the cell dissociation solution/cell suspension. Using a 10 mL serological pipette, add the cell suspension into a sterile 50 mL centrifuge tube. Make a pellet of cells using a centrifuge (150 &#215; g, 5 min, 22&#160;&#176;C).</li>
	<li>While the pellet is being formed, remove the gelatin solution from the 96-well plate via aspiration using a Pasteur pipette connected to a vacuum. Remove the plate lid from the time of gelatin removal until the solution is ready to be plated. 
	NOTE: A multichannel pipette or sterile manifold connected to a vacuum may optionally be used.</li>
	<li>Remove the medium from the centrifuge tube by aspiration using a Pasteur pipette connected to a vacuum or a serological pipette; be careful not to disturb the cell pellet.</li>
	<li>Add fresh DMEM complete medium to the centrifuge tube using a 5 or 10 mL serological pipette. Gently break up the cell pellet by mixing the DMEM complete medium with a 1,000 &#181;L pipette or a 5 mL serological pipette. 
	NOTE: Eight milliliters of DMEM are typically used, but higher volumes can be added to make cell counting easier.</li>
	<li>Mix 10 &#181;L of the cell suspension with 10 &#181;L of trypan blue in a microcentrifuge tube using a pipette with either 20 &#181;L or 200 &#181;L tips. Add 10 &#181;L of the cell suspension/trypan blue mixture to a hemocytometer and count the cells.</li>
	<li>Add DMEM complete media to the cell suspension to make a density of 600,000 cells/mL using either a 10 or 25 mL serological pipette. This will give a final density of 60,000 cells/well in a 96-well plate (determined to be the optimal density by experimentation). 
	NOTE: A normal successful cell culture will yield between 12,000,000 and 15,000,000 cells, which can seed two to three 96-well plates.</li>
	<li>Mix the cell suspension by gently inverting the centrifuge tube and add it to a 50 mL multi-channel pipette reservoir. Mixing the suspension in the reservoir every 30 s by gently rocking the reservoir side to side, add 100 &#181;L of the cell suspension to each well using a multi-channel pipette. Replace the transparent plate cover and gently shake the plate by sliding it on the surface of the sterile hood from North to South and East to West to ensure even cell distribution. Incubate the plate in the incubator (37 &#176;C, 5% CO2) for 16-24 h. 
	NOTE: Additional cell suspension can be used to prepare additional plates, or steps 1.10-1.11 can be repeated to continue the cell line.</li>
</ol>
3. Calcium mobilization assay preparation
<ol>
	<li>Prepare assay reagents.
	<ol>
		<li>Prepare probenecid solution (250 mM, aqueous) by adding 36 mg of probenecid to a microcentrifuge tube and dissolving it in 0.6 M NaOH (500 &#181;L). Vortex the solution. 
		NOTE: Probenecid improves the retention of dye in the cell by inhibiting organic ion transporters75,76,77.</li>
		<li>To make HBSS-HEPES assay buffer, add HEPES (1.19 g) to a 500 mL bottle of Ca/Mg/phenol red-free HBSS (making a 10 mM solution of HEPES). Supplement the buffer with MgCl2 (1 M aqueous solution, 500 &#181;L) and CaCl2 (1 M aqueous solution, 500 &#181;L). Mix the solution and store in a refrigerator at 5 &#176;C when not in use.
		<ol>
			<li>For each 96-well plate, add 50 mL of the HBSS-HEPES assay buffer to a 50 mL centrifuge tube, supplement with 500 &#181;L of the probenecid solution, and mix well. Warm the buffer to room temperature prior to use.</li>
		</ol>
		</li>
		<li>Make a 10% Pluronic F-127 solution in DMSO by adding Pluronic F-127 (20 mg) to a microcentrifuge tube or HPLC&#160;vial and dissolving it with 200 &#181;L of DMSO. 
		NOTE: This solution can be stored at room temperature and is sufficient for about 30 plates. Pluronic F-127 dissolves very slowly at room temperature and should be gently heated to facilitate dissolution.</li>
		<li>Prepare the dye loading buffer by first adding 24 &#181;L of DMSO to a vial of Fluo-4/AM (50 &#181;g) to dissolve the dye. In a foil-wrapped 15&#160;mL centrifuge tube, add 6 mL of HBSS-HEPES assay buffer and supplement with 6 &#181;L of 10% Pluronic F-127 and 6 &#181;L of Fluo-4/AM solution. Briefly vortex the solution and allow to sit at room temperature, out of light, for 10 min. 
		NOTE: To prevent photobleaching of Fluo-4, keep the dye out of light by wrapping aluminum foil around the centrifuge tube containing the dye solution and the lid of the assay plate.</li>
	</ol>
	</li>
	<li>Prepare assay plates.
	<ol>
		<li>Remove the assay plate from the incubator and confirm confluency using an inverted microscope. Manually remove the DMEM complete media by flicking it into a sink and blotting the top of the plate with a paper towel. 
		NOTE: Medium is sufficiently removed by holding the plate horizontally and flicking, followed by rotating the plate 180&#176;and repeating.</li>
		<li>Add HBSS-HEPES assay buffer plus probenecid solution to a 50 mL multi-channel pipette solvent reservoir. 
		NOTE: This solution will be used for later washing steps and should be put aside and covered with aluminum foil until needed.</li>
		<li>Add 100 &#181;L of the HBSS-HEPES assay buffer plus probenecid solution to each well with a multi-channel pipette and allow the cells to sit in the presence of probenecid for 5 min. Remove the HBSS-HEPES assay buffer by flicking into the sink in the same manner as in step 3.2.1.</li>
		<li>Add dye loading buffer to a separate 50 mL multi-channel pipette solvent reservoir. Add 50 &#181;L of the dye loading buffer to each well of the assay plate using a multi-channel pipette. Incubate the plate for 45 min (37 &#176;C, 5% CO2).</li>
		<li>While the plate is in the incubator, prepare the agonist/antagonist solutions.
		<ol>
			<li>Antagonist solutions are stored as 31.6 mM solutions in DMSO. In a PCR tube, dilute 2 &#181;L of the stock solution with 38 &#181;L of 0.1% BSA/water to obtain a 1.58 mM solution. 2 &#181;L of this solution will give a final concentration of 31.6 &#181;M in the assay plate. Dilute 4 &#181;L of the 1.58 mM antagonist solution with 36 &#181;L of 5% DMSO/water (no BSA addition), and prepare the remaining concentrations through serial dilutions.</li>
			<li>Prepare a 108.3 &#181;M (16.7x) solution of TFLLRN-NH2 in HBSS-HEPES assay buffer, which will give a final concentration of 6.5 &#181;M when 6 &#181;L is added to each well of the assay plate. For example, dissolve a 2 mg aliquot of TFLLRN-NH2 with 5.244 mL of HBSS-HEPES assay buffer to give a 0.5 mM stock solution. Mix 1.083 mL of this solution with 3.917 mL of HBSS-HEPES assay buffer to give a 108.3 &#181;M solution. 
			NOTE: TFLLRN-NH2 solutions should be stored in a -20 &#176;C freezer when not in use.</li>
		</ol>
		</li>
		<li>Remove the plate from the incubator and ensure dye uptake by using a fluorescence microscope at a suitable setting (typically for green fluorescent protein, see Figure 1 for an example of Fluo-4 successfully loaded into cells). Remove the dye loading buffer by flicking the plate in the same manner as step 3.2.1.</li>
		<li>Wash the cells 2x with 50 &#181;L of the HBSS-HEPES assay buffer plus probenecid solution (from step 3.2.3) in each well (remove by flicking medium into the sink).</li>
		<li>Add 92 &#181;L of HBSS-HEPES assay buffer to each well and again view the cells with a fluorescence microscope to ensure excess dye has been fully removed, and that cells remain adherent.</li>
		<li>Use a multi-channel pipette to add 2 &#181;L of the antagonist solutions and vehicle to appropriate wells (see Table 1 for a representative plate map).</li>
	</ol>
	</li>
</ol>
4. Performing the calcium mobilization assay
<ol>
	<li>Set the plate reader as follows: temperature: 37 &#176;C; excitation wavelength: 485 nm; emission wavelength: 525 nm; measurement height: 7.8 mm (optimized); number of flashes: 100; number of repeats: 20.</li>
	<li>Incubate the plate for 15 min in the plate reader at 37 &#176;C. Measure the background fluorescence in columns 1 and 2 (five scans per well).</li>
	<li>Eject the plate from the plate reader and quickly add 6 &#181;L of agonist solution to each well in columns 1 and 2 from an 8-tube PCR strip using a multi-channel pipette. 
	NOTE: For this step, we use an electronic 8-channel pipette with 0.1-10 &#181;L pipette tips.</li>
	<li>Measure the change in fluorescence as calcium mobilization occurs in the cells. Perform 20 scans of each well according to the settings above. 
	NOTE: Scanning each well in two columns 20x each takes approximately 5 min (i.e., ~15 s between scans of each well).</li>
	<li>Repeat steps 4.2-4.4 for columns 3-12 in two column increments.</li>
</ol>
5. Data analysis
<ol>
	<li>Export data from the assay as separate spreadsheets for each two-column group.</li>
	<li>Find the average of the basal fluorescence readings by using the function AVG(first scan: last scan). Do this for every well in both columns.</li>
	<li>Find the maximum fluorescence after the addition of agonist by using the function MAX(first scan: last scan).</li>
	<li>Calculate the change in fluorescence by subtracting the basal fluorescence from the maximum fluorescence.</li>
	<li>Subtract the change in fluorescence calculated for the negative control (addition of vehicle without agonist) from each well in the same column.</li>
	<li>Find the relative (normalized) change in fluorescence by dividing the change in fluorescence in each well by the change in fluorescence in the vehicle (0.1% DMSO/water) + 6.5 &#181;M TFLLRN-NH2 well.</li>
	<li>Copy the non-control values, as percentages, into a statistics and graphing software program. If using the referenced software, choose the XY table option, with the Numbers chosen as the X-axis and replicate values in side-by-side subcolumns as the Y-axis.</li>
	<li>Use the program to plot concentration-response curves (CRCs) from the data. If using the referenced software, choose the log(inhibitor) vs. response - Variable slope (four parameters) option.</li>
</ol>

Studying Protease-Activated Receptor Ligands in Endothelial Cells

C.D. is inventor on patents involving parmodulins and is the founder and a shareholder of Function Therapeutics, Inc., which is developing parmodulins for clinical use.

While the previously reported protocol72 was generally reliable and allowed us to identify a new lead parmodulin, NRD-21,62 a more efficient protocol was desired. The assay was further compromised during the supply shortage caused by the COVID-19 pandemic. Acquiring tips for the automated liquid handler became difficult, and attempting to wash, sterilize, and reuse the tips produced CRCs with significant variance. This facilitated an urgent series of experiments designed to...

Protease-activated receptors (PARs)1,2,3 are a subfamily of class A G protein-coupled receptors (GPCRs) that are expressed in a variety of cell types, including platelets and endothelial cells4,5,6,7. Unlike the majority of GPCRs, PARs have a unique intramolecular mode of activation. Most GPCRs are activated by soluble ligands interacting with a distinct binding pocket, but PARs are activated by the proteolytic cleavage of the N-terminus, which results in a new tethered ligand that can interact with the extracellular loop 2 domain on the surface of a cell6,8,9. This interaction activates the receptor and can initiate several signaling pathways, promoting effects such as inflammation and platelet activation4,10,11,12. Different proteases can activate PARs through cleavage at unique sites on the N-terminus, revealing different tethered ligands (TL) that stabilize receptor conformations, which initiate different signaling pathways9,13,14,15. For example, in the most well-studied member of the subfamily, PAR1, cleavage by thrombin is used to support numerous biological processes, including platelet activation and leukocyte recruitment to the endothelium, but can lead to deleterious effects when the receptor is overexpressed or overactivated4,16,17,18,19,20,21. Conversely, cleavage by activated protein C (aPC) can promote anti-inflammatory effects and maintenance of endothelial barriers15,22,23,24,25,26,27,28,29. PARs can also be activated by peptide analogs of the TLs in an intermolecular fashion13,30,31. These peptides are routinely used to measure PAR inhibition (modulation) in place of PAR-targeting proteases, and they are used in this protocol.
Numerous disorders are associated with pathological PAR1 signaling, including sepsis22,32, cardiovascular disease33,34,35,36,37,38, kidney disease39,40,41,42, sickle cell disease43, fibrosis44, osteoporosis and osteoarthritis45,46, neurodegeneration47,48,49,50,51, and cancer52,53,54,55,56,57,58,59. Antagonists of PAR1 have been studied since the 1990s as antiplatelet agents for cardiovascular disease, and the growing list of diseases associated with the receptor necessitates the identification of novel ligands for use as biological probes (tool compounds) or as potential therapeutics. Currently, there is only one FDA-approved PAR1 antagonist, vorapaxar, which is used to treat coronary artery disease in high-risk patients34,36,37,60. An alternative PAR1 antagonist, the pepducin PZ-128, completed a successful phase II study to prevent thrombosis in cardiac catheterization patients38. The Dockendorff group has focused on the medicinal chemistry and pharmacology of a separate class of small molecules, PAR1 ligands known as parmodulins61,62. Unlike reported PAR1 antagonists such as vorapaxar, parmodulins are allosteric, biased modulators of PAR1 that selectively block the G&#945;q pathway while promoting cytoprotective effects similar to aPC. Unlike potent orthosteric PAR1 antagonists such as vorapaxar, published parmodulins are also reversible63,64,65.
Initially, parmodulins were identified by Flaumenhaft and coworkers for&#160;their ability to inhibit P-selectin expression or granule secretion in platelets61,66. However, an alternative method was required to study the effects of parmodulins on endothelial cells. One common method to monitor GPCR-related signaling is to measure intracellular Ca2+ mobilization, an important secondary messenger that can be measured using a suitable intracellular calcium-binding dye67,68. Substantial evidence has been provided showing that calcium mobilization induced by PAR1 is through the activation of G&#945;q69,70. Once activated by its tethered ligand (or a suitable exogenous ligand), PAR1 undergoes a conformational change which causes guanosine diphosphate (GDP) bound to the G&#945;q subunit to be replaced by guanosine triphosphate (GTP)68. The G&#945;q subunit then activates phospholipase C&#946; (PLC-&#946;), which catalyzes the hydrolysis of phosphatidylinositol 4,5 bisphosphate (PIP2), forming 1,4,5-inositol triphosphate (IP3) and diacylglycerol (DAG). Finally, IP3 binds to IP3-sensitive Ca2+ channels in the membrane of the endoplasmic reticulum, allowing Ca2+ to be released into the cytoplasm, where it can bind to Ca2+-dependent fluorescent dyes, such as Fluo-4, that are added to the cells71.&#160;This process occurs within seconds and can increase the concentration of Ca2+ 100-fold, leading to a drastic change in the amount of calcium-bound dye and a robust fluorescence signal.
In 2018, the Dockendorff group disclosed a medium-throughput Ca2+ mobilization assay that could be used to identify antagonists of the G&#945;q pathway of PAR172. The assay used&#160;EA.hy92673, a hybrid human endothelial cell line, which can be used for multiple passages without a noticeable change in PAR1 expression, and is established for in vitro measurements of cytoprotective effects.&#160;
The original protocol used EA.hy926 cells in 96-well plates and loaded with Fluo-4/AM dye, which was chosen due to its intense fluorescence at 488 nm and high cell permeability. Once the dye was loaded into the cells, lengthy washing steps were performed with an automated 8-channel liquid handler (faster methods of liquid handling, such as a 96-channel washer, were inaccessible). The reproducibility of this assay was superior to that without the careful, automated robotic media changes. Antagonists were then incubated with the cells, PAR1 was activated through the sequential addition of a selective agonist (16 wells at a time), and changes in fluorescence resulting from calcium mobilization and dye binding were measured to determine activity.
While this protocol allows for the measurement of PAR1-mediated calcium mobilization, it is limited by the time required to assay each 96-well plate. Long experiment times are problematic not only because the number of compounds that can be screened each day is limited, but also because dye efflux occurs over time, narrowing the assay window by increasing the basal fluorescence. One contributor to the long experiment time is the use of an 8-channel liquid handler for plate washing, which adds over 30 min to each experiment. The required tips also became difficult to obtain due to supply chain problems. Here an updated protocol for the PAR-mediated calcium mobilization assay that does not require a liquid handler, and therefore can be run in higher throughput, is reported. This protocol should also be suitable for measuring signaling with other GPCRs that lead to intracellular calcium mobilization.&#160;This updated plate reader protocol&#160;is ideal for academic and small industrial labs that do not have the resources for expensive cell imaging instruments but have a need to rapidly screen numerous compounds. For an example of a calcium mobilization assay using a plate imager, see Caers et al.74.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Culture Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adherent EA.hy926 cells</td>
			<td>ATCC</td>
			<td>CRL-2922</td>
			<td></td>
		</tr>
		<tr>
			<td>CellStripper cell dissociation reagent</td>
			<td>Corning</td>
			<td>25-056-CI</td>
			<td>Trypsin can optionally be used, but should definitely be avoided with PAR2 assays.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) w/phenol red</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Avantor</td>
			<td>97068-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>MilliporeSigma</td>
			<td>G2500</td>
			<td>Use to make an aqueous 0.4% (w/v) solution with deionized water. Autoclave before use to sterilize.</td>
		</tr>
		<tr>
			<td>Pen/Strep (100X)</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue (0.4% w/v)</td>
			<td>Corning</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Mobilization Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Thermo</td>
			<td>172571000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Avantor</td>
			<td>97061-420</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Thermo</td>
			<td>42352-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Thermo</td>
			<td>J66650-AD</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4/AM</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (Ca/Mg/phenol-red free)</td>
			<td>Corning</td>
			<td>21-022-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>MilliporeSigma</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127 (Poloxamer 407)</td>
			<td>Spectrum Chemical</td>
			<td>P1166</td>
			<td></td>
		</tr>
		<tr>
			<td>Probenecid</td>
			<td>TCI America</td>
			<td>P1975</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>VWR International</td>
			<td>BDH9292</td>
			<td></td>
		</tr>
		<tr>
			<td>TFLLRN-NH2 (TFA salt)</td>
			<td></td>
			<td></td>
			<td>Prepared by Trudy Holyst at the Versiti Blood Research Institute</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture-treated, black-walled, clear bottom assay plate</td>
			<td>Corning</td>
			<td>3603</td>
			<td>with transparent lids</td>
		</tr>
		<tr>
			<td>Centrifuge tube, 15 mL</td>
			<td>Avantor</td>
			<td>89039-664</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube, 50 mL</td>
			<td>Avantor</td>
			<td>89039-656</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture flask, T-75</td>
			<td>Corning</td>
			<td>353136</td>
			<td>tissue culture treated</td>
		</tr>
		<tr>
			<td>Disposable reagent reservoir, 50 mL</td>
			<td>Corning</td>
			<td>RES-V-50-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Enspire plate reader</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Discontinued</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube, 1.5 mL</td>
			<td>Avantor</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette, 9&#34;</td>
			<td>Fisher</td>
			<td>13-678-6B</td>
			<td>must be sterilized</td>
		</tr>
		<tr>
			<td>PCR tube strip with separate flat cap strips</td>
			<td>Avantor</td>
			<td>76318-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips, 20 &#181;L</td>
			<td>Biotix</td>
			<td>63300042</td>
			<td>sterile, filtered tips</td>
		</tr>
		<tr>
			<td>Pipette tips, 200 &#181;L</td>
			<td>Biotix</td>
			<td>63300044</td>
			<td>sterile, filtered tips</td>
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			<td>Pipette tips, 1250 &#181;L</td>
			<td>Biotix</td>
			<td>63300047</td>
			<td>sterile, filtered tips</td>
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			<td>Prism</td>
			<td>GraphPad</td>
			<td></td>
			<td>volume 6 used</td>
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			<td>Serological pipette, 5 mL</td>
			<td>Tradewinds Direct</td>
			<td>&#160;07-5005</td>
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			<td>Serological pipette, 10 mL</td>
			<td>Tradewinds Direct</td>
			<td>&#160;07-5010</td>
			<td></td>
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		<tr>
			<td>Serological pipette, 25 mL</td>
			<td>Tradewinds Direct</td>
			<td>&#160;07-5025</td>
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characterizing modulators of protease-activated receptors with a calcium mobilization assay using a plate reader

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of G&#945;q signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.

Author Spotlight: Developing Parmodulins to Target Protease-Activated Receptors for Inflammation Control

Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader

Our lab is working to understand how the modulation of protease activated receptors can inhibit inflammation. We have developed a new class of small molecules called parmodulins that modulate the activity of PAR1. The goal is to discover parmodulins with improved properties for potential use as drug candidates, and also to understand their mode of action.In preclinical studies with our collaborators, we've determined that parmodulins may be particularly promising for the treatment of disorders driven by thromboinflammation, including kidney, liver, and cardiovascular disease. Their mode of action may also enable the development of compounds with a better safety profile than prior PAR1 targeting drug candidates. The receptor PAR1 is important for activating platelets, but it's signaling in endothelial cells is also important and relevant in numerous pathologies.Therefore, we wanted to develop a cheap and convenient protocol for testing the activity of parmodulins and cultured endothelial cells. We're here to share an updated protocol measuring calcium mobilization that uses a standard plate reader, and does not require an automated liquid handler. It's suitable for screening PAR1 ligands in endothelial cells, but in theory, it can be used to measure the activity of any compounds affecting calcium mobilization.

The purpose of this assay is generally to produce concentration-response curves (CRCs) for three to four new parmodulins. On each assay plate, an additional CRC for a known compound, such as NRD-21, is often generated that acts as a quality check for the experiment due to its known IC50. To generate CRCs, a plate map such as the one depicted in Table 1 should be planned. If single-point concentration-responses are desired instead, compounds at 10 &#181;M final concentrations (or other preferre...

An improved protocol for a calcium mobilization assay with endothelial cells, used to identify ligands of protease-activated receptors (PARs), has been developed. The new protocol reduces total assay time by 90-120 min and yields reproducible concentration-response curves.

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding ...

characterizing-modulators-protease-activated-receptors-with-calcium

Research

JoVE Journal

Biochemistry

283 Views.  Function Therapeutics. Our lab is working to understand how the modulation of protease activated receptors can inhibit inflammation. We have developed a new class of small molecules called parmodulins that modulate the activity of PAR1. The goal is to discover parmodulins with improved properties for potential use as drug candidates, and also to understand their mode of action.In preclinical studies with our collaborators, we've determined that parmodulins may be particularly promising for the treatment of d...

Video: Author Spotlight: Developing Parmodulins to Target Protease-Activated Receptors for Inflammation Control

Culturing the Endothelial Cell Line in 96-Well Plates for the Calcium Mobilization Assay

To begin, arrange the required experimental materials on the working platform. Add 100 microliters of sterile 0.4%gelatin solution to the wells of a black walled 96 well clear bottom plate. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 30 minutes.Under an inverted microscope, confirm that endothelial cells in a T75 flask are 80 to 100%confluent. Remove the DMEM complete medium from the flask via aspiration. To wash the cells, add 10 milliliters of PBS and gently shake the flask.Replace the PBS with five milliliters of cell dissociation solution. Incubate the flask at 37 degrees Celsius and 5%carbon dioxide for approximately 12 minutes. Tap the flask after six minutes.To help facilitate dissociation. Add five milliliters of the prewarmed DMEM complete medium to the flask, and mix the cell suspension. Using a 10 milliliter serological pipette, transfer the cell suspension into a sterile 50 milliliter tube and centrifuge at 150 G for five minutes.In the meantime, using a pastor pipette connected to a vacuum, aspirate the gelatin solution from the 96 well plate. After centrifugation of the cells, remove the supernatin from the tube without disturbing the pellet. Using a serological pipette, add eight milliliters of fresh DMEM complete medium to the tube, and gently pipette the suspension to break up the cell pellet.Count the cells using trippen blue on a hemocytometer. Add DMEM complete medium to make a density of 600, 000 cells per milliliter suspension. Mix the cell suspension by gently inverting the tube.Add the cell suspension to a 50 milliliter multi-channel pipette reservoir. Every 30 seconds, gently rock the reservoir side to side to mix the suspension and use a multi-channel pipette to add 100 microliters to each well of the 96 well plate coated with gelatin. Replace the transparent plate cover and gently shake the plate by sliding it on the surface of the sterile hood from north to south and east to west.Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for 16 to 24 hours.

Enhanced Plate Reader Assay for Calcium Mobilization in Endothelial Cells

After growing endothelial cells in the 96 well plate, remove it from the incubator and confirm confluency using an inverted microscope. Flick the plate into a sink to remove the complete media, and blot the top of the plate with a paper towel. Add HBSS HEPES assay buffer plus probenecid solution to a 50 milliliter multi-channel pipette solvent reservoir.Using a multi-channel pipette, add 100 microliters of assay buffer to each well and allow the cells to sit for five minutes. Then, flick the plate to remove the assay buffer. Add downloading buffer to a separate 50 milliliter multi-channel pipette solvent reservoir.Using a multi-channel pipette, add 50 microliters of the dye-loading buffer to each well of the assay plate. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 45 minutes. After incubation, observe the plate under a fluorescence microscope to confirm the dye uptake.Flick the plate to remove the dye solution. Wash the cells twice with 50 microliters of the HBSS HEPES assay buffer plus probenecid solution in each well. Flick the plate to remove the assay buffer.Add 92 microliters of HBSS HEPES assay buffer to each well, and again, view the cells with a fluorescence microscope to ensure excess dye has been fully removed and that cells remain adherent. Using a multi-channel pipette, add two microliters of the antagonist solutions and vehicle to appropriate wells. After turning on the plate reader, set the temperature to 37 degrees Celsius and incubate the plate for 15 minutes.Measure the background fluorescence in columns one and two, performing five scans per well. Eject the plate from the plate reader, then from an 8-tube PCR strip, quickly add six microliters of agonist solution using a multi-channel pipette to each well in columns one and two. Measure the change in fluorescence as calcium mobilization occurs in the cells.Perform 20 scans of each well. The calcium mobilization assay with endothelial cells demonstrated that positive control wells with no antagonist exhibited a rapid increase in fluorescence. Wells with high antagonist concentrations showed minimal change in fluorescence, similar to the negative control wells with no agonist.