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 parmamodlins, that modulate the activity of Par 1. The goal is to discover parmadolins 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 parmadolins may be particularly promising for the treatment of disorders driven by thrombo inflammation, including kidney, liver, and cardiovascular disease. Their mode of action may also enable the development of compounds with a better safety profile than prior Par 1 targeting drug candidates. The receptor Par 1 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 parmadolins, 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 Par 1 ligands in endothelial cells, but in theory, it can be used to measure the activity of any compounds affecting calcium mobilization.

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 pre-warm 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 supernatant from the tube without disturbing the pellet. Using a serological pipette, add eight milliliters of fresh DMEM complete medium to the tube, and gently pipet the suspension to break up the cell pellet. Count the cells using trippen blue on the 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 multichannel pipette reservoir.

Every 30 seconds, gently rock the reservoir side to side to mix the suspension, and use a multichannel 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.

After growing endothelial cells in the 96 well plate, remove it from the incubator, and confirm co fluency, 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 Heaps Assay Buffer Plus Pro Benefit Solution to a 50 milliliter multichannel 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 Heaps Assay Buffer Plus Pro Benefit Solution in each well. Flick the plate to remove the assay buffer.

Add 92 microliters of HBSS Heaps 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 multichannel 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-tubes 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.