GPCRs (G protein-coupled receptors)

Cardiomyocyte

Early prediction of drug-induced functional cardiotoxicity requires robust in vitro systems suitable for high-throughput screening. Readily-available iPSC-derived human cardiomyocytes may be used in conjunction with calcium-sensitive dyes, with beat rates and patterns monitored aschanges in intracellular calcium in response to both GPCRs and ion channels. These calcium peaks can be analyzed with our ScreenWorks Peak Pro 2 software which provides the investigator a suite of powerful tools for quantification of cardiomyocyte activity.

Read application notes:

• Compound effects upon calcium transients in Cor.4U human iPSC cardiomyocytes
• High throughput cardiotoxicity assays using stem cell-derived cardiomyocytes

Cardiotoxicity: calcium oscillations

Induced pluripotent stem cell (iPSC)- derived cardiomyocytes are a particularly attractive in vitro model system due to their use for evaluation of compound effects on both cardiac function and safety. Calcium signal oscillation reflects changes in cytoplasmic calcium concentration making it possible to use a calcium sensitive dye such as the EarlyTox^™ Cardiotoxicity Kit.

Read application notes:

• Measure Calcium Oscillation in iCell Cardiomyocytes2 on the FLIPR Tetra System
• Multi-parametric assessment of compound-induced pro-arrhythmic effects in human iPSC-derived cardiomyocytes

Evaluation of calcium flux

The evaluation of calcium flux is a long accepted, tried and true measure of cellular activity. Calcium flux can be used as a measurement for a host of cellular processes including neurotransmitter release, GPCR activity, ligand gated ion channels, and cardiomyocyte beat patterns, among many others. The FLIPR System is the drug discovery tool for evaluation of calcium flux in high and ultra-high throughput screening due to its ease of use, sensitivity and user configurability.

Learn more:

• Enhance your calcium screens with FLIPR Calcium 6 Assay Kits
• Comparison of a novel calcium assay to fluorescence-based calcium flux assays

Fura-2 QBT calcium

Fura-2 dye has long been considered an important tool to measure calcium mobilization in cellular imaging, GPCR mediated intracellular calcium flux, and ion channel activation. This ratiometric dye helps correct for assay inconsistencies in dye loading or cell plating through calculating the fluorescence intensity ratio between bound and free indicators. However washing is required, increasing well-to-well variability and adding time and complexity to each assay.

The Fura-2 QBT™ Calcium Kit from Molecular Devices incorporates proven quench based technology with a ratiometric Fura-2 calcium indicator to provide a homogenous assay to minimize cell based variability, while increasing throughput through elimination of cell washing prior to detection.

Learn more:

• Fura-2 QBT Calcium Kit: A homogenous Fura-2 calcium assay
• Fura-2 QBT ratiometric calcium kit for FLIPR Tetra System or Flexstation 3 reader

Homogeneous solution for GPCR assays

Cell-based assays have become an indispensable method for screening and compound profiling in the early drug discovery process. To date, such assays have proven to be some of the most reliable and reproducible methods in receptor characterization studies, primary screening campaigns and compound profiling programs. For Gq-coupled GPCR targets specifically, homogeneous fluorescent calcium flux assays with masking technology are the methodology of choice.

Combining a novel fluorophore and proven masking technology, the FLIPR Calcium 5 Assay Kit delivers reliable pharmacology, a larger signal window, and improved assay performance. With the FLIPR Calcium 5 Assay Kit and the FLIPR System, consistent screening of a variety of receptors and targets, especially those with small calcium signal responses, can be obtained in an easy-to-use, homogeneous format.

Read Application Note:

• Homogeneous solution for GPCR assays with FLIPR Calcium 5 Assay Kit

Live cell Gi - and Gs-coupled GPCR second messenger signaling

Detection of G_i and G_s -coupled GPCR second messenger signal activity has been traditionally accomplished using assays such as radioactive binding or endpoint cAMP assays that require cell lysis. Such assays measure activity at a single time point in the cellular response and do not provide kinetic information. Another option utilizes forced-coupling of G_i and G_s -coupled GPCRs to Gα16 followed by fluorescence detection of calcium flux upon agonist receptor activation. Again, this assay is sub-optimal as it does not signal through the biorelevant cAMP pathway.

Here we demonstrate endogenous receptor activity in CHO-K1 and HEK-293 cell lines stably expressing the GloSensor^™ plasmid using the FLIPR System.

Read Application Note:

• Live cell Gi- and Gs -coupled GPCR second messenger signaling on the FLIPR Tetra System

Live cell kinetic assays

The ImageXpress Confocal and Micro 4 High-Content Imaging Systems offer optional fluidics and environmental control modules, enabling single-channel pipetting for compound addition and media exchange during rapid kinetics or extended time-lapse experiments. The Transfluor Cell-Based GPCR Assay Kit is a valuable tool used during high content screening (HCS) to track G-protein coupled receptor (GPCR) activation by quantifying GPCR desensitization and recycling using GFP-labeled ß-arrestin with automated image analysis.

Read Application Note:

• Live cell kinetics assay utilizing the ImageXpress Micro System

Measure intracellular calcium flux

G protein-coupled receptors (GPCRs) play an important role in cell signaling. When the receptor is activated by a ligand, receptor conformation is changed, triggering G-protein activation inside the cell. An active G protein has the potential to induce various cascades of intracellular messengers including calcium. The FLIPR System performs high-throughput, functional cell-based assays and is the system of choice in drug discovery for evaluating changes in intracellular calcium detected through use of fluorescent calcium-sensitive reporter dyes. Here we provide a basic protocol for performing a calcium mobilization assay on the FLIPR System using the FLIPR® Calcium Assay Kit, a homogeneous, fast and reliable fluorescence assay.

Read Application Note:

• Comparison of intracellular calcium measurements with FLIPR Calcium Assay Kit

Monitoring Gq-coupled protein receptor activity

Gq protein-coupled receptor activation is commonly monitored in live cells in real time using calcium-sensitive dyes on a fluorescence plate reader. Automated liquid handling within the plate reader is generally required to deliver agonist compounds to the cells in the microplate while the detection system takes real-time readings of compound-induced changes in fluorescence intensity values. Analysis of the resulting kinetic readings yields information about the compound response profiles, including EC₅₀ and IC₅₀ values for agonists and antagonists.

Read Application Note:

• Monitoring Gq protein-coupled receptor activation on the SpectraMax i3x reader with injector module

Neuronal assays

The FLIPR System is an excellent tool for monitoring calcium flux related to neuronal activity. Newly developed commercially available iPSC neurons have made high-throughput neuronal screening a reality. These cells and assays are essential for monitoring a variety of factors including neural cell culture conditions, drug compound effects, and the effect of environmental neurotoxins. Neuronal activity and neurotransmitter release can be evaluated with fluorescence-based calcium indicator dyes combining fast, simple, and powerful assays with scalable throughput.

Learn more:

• Neurotoxicity profiling using 3D human iPSC-derived neural spheroids
• Phenotypic characterization of neuroactive compound effects
• Multiplexed assays for neurotoxicity evaluation using neural 3D cell models
• Complex event analysis for neurotoxic profiling of compound effects on human iPSC-derived neural spheroid 3D cultures