culturing the endothelial cell line in 96-well plates for the calcium mobilization assay

Studying Protease-Activated Receptor Ligands in Endothelial Cells

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.

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.

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.

culturing-endothelial-cell-line-96-well-plates-for-calcium

Research

JoVE Journal

Biochemistry

22 Views. 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 sha...