FLIPR Calcium Assay Kits – A Reliable Cellular Assay Screening System

G-protein-coupled receptors (GPCRs) and ion channels that control calcium levels are prime targets for HTS drug discovery. Researchers employ a calcium-sensitive dye to efficiently monitor changes in intracellular Ca2+ flux and screen large compound libraries.

FLIPR assay do typically involve multiple steps of dye loading and washing cells, which can increase sample handling variability. The FLIPR assay method utilises no-wash dye for high-throughput calcium monitoring in FlexStation HTS systems.

An argon-ion laser is used by Flipr to excite indicator dyes like Fluo-3. This keeps cells from being exposed to UV light, which could be harmful, and lets FLIPR Tetra use expensive biochemical assay reagents without affecting performance.

FLIPR requires optimising its experimental setup to achieve accurate, repeatable results, which involves setting up an appropriate sequence with sufficient addition volumes and selecting suitable filters for every plate.

Scalability

The FLIPR system provides an accurate and efficient real-time kinetic cellular assay screening solution, particularly during the early drug discovery stages. You can configure it with LED sets, filters, and luminescence or fluorescence detection according to your assay needs; additionally, it supports 96-well plates as well as various pipettor heads to perform no-wash and cell suspension assay formats.

This system has been optimised to perform fluorescent and luminescent assays such as aequorin for GPCR screening and ion channel screening, making it the screening solution of choice in many laboratories. Furthermore, Molecular Devices also offers homogeneous no-wash assay kits designed specifically for FLIPR, which measure calcium flux and membrane potential in order to measure their potential.

The FLIPR system can improve scalability and performance by running multiple assays simultaneously using multiple pipettor heads in parallel, increasing assay throughput by over twofold compared to traditional methods. Its in-tip dilution feature also cuts down on turnaround times by a large amount and lets higher buffer concentrations work, which raises sensitivity while lowering background signal at the same time.

Reliability

Flipr manages millions of changes at scale, so its reliability must be secure. We use a fan-out cache model that is eventually consistent in order to ensure clients can keep working if something goes wrong with gateways, backends, or networks by reading from disk. Host agents then pull data from gateways and store it on discs in a way clients can read.

The FLIPR Penta system utilises a high-speed camera option in combination with ScreenWorks Peak Pro 2 software to achieve faster frame resolution and improved detection of key dynamics for GPCRs and ion channels. Its high-resolution capability enables more precise monitoring of membrane potential as well as data that corresponds with manual patch clamp assay results, making it an invaluable resource for uncovering subtle kinetic responses from these proteins.

Getting rid of compounds that cause false-positives or false-negatives in an inhibitor or agonist screening process is one of the hardest parts of a high-throughput FLIPR screen. Fluorescent compounds, Ca2+ ionophores and compounds that permeabilize cell membranes may all skew results; it’s difficult for one individual to do this alone and requires expert review of response data for identification. To get around this problem, Molecular Devices has a number of FLIPR membrane potential assay kits that are designed to get the best indicator dye/quencher combinations and the ability to work with different cell lines, channels, and compounds so that accurate screening can happen without any unwanted compounds.

Security

Flip collects and processes data in order to provide, enhance, and develop its service, meet customer and technical service requests, and comply with applicable laws. In order to protect your privacy, Flip employs best practices and standards for data security, such as encryption in transit and at rest, as well as manual and automated monitoring and audit systems, as well as access control measures.

We amplified the FLIPr gene using primers (5′-ACTGCGGGATCCTTTTTTAGCTAATGG-3′; BamHI site underlined) integrated into pLipo. PCR products were cloned into the BamHI and XhoI sites of pLipo to form pLF.

Host-agents, part of the fan-out cache, are responsible for pulling configuration from gateways and persisting it on disc for later retrieval by services. One major benefit is that in case gateways, backends, or networks go down, services can still work by reading off of the disc.

A peer must review and approve all configuration changes before deploying them to production environments. Flip also deploys changes incrementally across physical dimensions, minimising any unintended consequences that might occur from sudden updates. Monitoring systems allow rolled-back configuration if any problems arise as a result of changes made, all of which combine to ensure Flip remains a safe, reliable, and secure system.

Molecular Devices offers a variety of calcium assay kits to meet your research needs.

The FLIPR GPCR and calcium assay kits come with homogeneous fluorescence formulations that are already made. This makes it easy to quickly and easily develop and screen assays. FLIPR calcium assays can be run using various instrument types, including fluorescent microscopes, high-throughput screening-capable plate readers, and flow cytometers.

In regular FLIPR calcium tests, membrane-permeable dyes bind to Ca2+ in cells and make their fluorescence more bright, which lets FLIPR find them. Unfortunately, this approach has a number of drawbacks: large downward spikes during liquid transfer may mask an agonist response, while cleaning dye-loaded cells requires additional time and reagents.

Molecular Devices has created an innovative assay chemistry to overcome these limitations by offering the highest-performing ratiometric FLIPR calcium assay available for GPCR and calcium channel targets. The FLIPR Calcium 6 Assay Kit features a new-generation calcium indicator dye with a proprietary formulation to provide an expanded assay window capable of dealing with difficult cell lines and receptors.

The FLIPR Calcium 3 Assay Kit is ideal for measuring GPCRs that signal through heterotrimeric G proteins such as Gai, Gaq, or Ga16. The assay can also be used to screen high-throughput samples such as 96 or 384-well plates for GPCR targets and ion channel targets that signal via these heterotrimeric G proteins and uses a non-toxic and highly soluble probe that measures inhibition of GTP binding by drug candidates or compounds that affect phospholipase C/Ca2+ activity within cultured cells.

The FLIPR Calcium 4 Assay Kit is ideal for high-throughput screening.

The FLIPR Calcium 4 Assay Kit enables high-throughput screening (HTS) of GPCRs and ion channels that regulate intracellular calcium. A unique fluorescent calcium indicator dye is used by the kit to detect intracellular Ca2+ flux induced by an agonist in standard HBSS cell assays. This high-tech dye combines fluorophore technology with better masking and quenching technologies. It greatly expands the assay signal window and makes it easier to detect compounds at the same time.

Assaying GPCRs requires cells stably expressing them and a fluorescent calcium sensor, as well as loading cells with a soluble form of calcium indicator dye and adding a calcium-sensitive buffer. When activated by an agonist known for inducing large intracellular Ca2+ responses, this response can be detected using fluorescence imaging plate reader technology.

The FLIPR system from Molecular Devices can find both direct and allosteric modulators of GPCRs by watching how calcium levels change inside cells. Usually run as a multiple-addition screen with a pre-incubation phase to allow compounds to bind, followed by a low-concentration addition of known agonists to detect potentiators, this screening provides accurate results.

An antagonism assay uses high concentrations of known antagonists to isolate inhibitors and identify them through measurement. Usually, this process requires iterative efforts with assistance from an assay expert in reviewing the data and distinguishing false positives from true negatives.

The FLIPR Calcium 3 Assay Kit enables low-throughput screening.

The FLIPR Calcium 3 Assay Kit is a useful tool for keeping an eye on changes in intracellular calcium using the FLIPR fluorometric imaging plate reader system. We specifically tailored all reagents for the FLIPR platform to provide pre-optimised, homogeneous assay formulations that facilitate screening GPCR and ion channel targets more quickly.

Conventional calcium assays use membrane-permeable dyes to bind Ca2+ in cells and increase fluorescence, making these assays difficult to set up and run with careful interpretation of kinetic traces to detect an agonist or inhibitor response. Furthermore, large downward spikes during liquid transfer can obscure an agonist response, while washing dye-loaded cells requires time and additional reagents.

This FLIPR-optimised, no-wash calcium assay kit removes these limitations. We specially formulated the reagents to minimise background fluorescence associated with cell-based calcium mobilisation assays and enhance signal-to-noise ratios. Additionally, we added quenching dye to further decrease background noise and improve assay sensitivity in FLIPR plate readers.

As well as offering an assortment of FLIPR-specific reagents, this assay features an intuitive sequence setup that enables users to define all liquid handling steps as well as any automated wash sequences, tip unloads, or sample transfer steps in their experiment by simply clicking green circles next to each step and making changes accordingly. By customising their FLIPR experiment to their individual needs by activating or deactivating settings using clickable green circles beside each step, users can customise their FLIPR experiment according to their individual needs.

The FLIPR Calcium 2 Assay Kit is ideal for endogenous or primary targets.

The FLIPR instrument allows high-throughput screening of GPCRs and ion channels that respond to changes in intracellular calcium using membrane-permeable dyes and fluorescence imaging plate reader technology. A cooled CCD camera imaging system simultaneously reads fluorescence signals from each well in the microplate at subsecond intervals to capture the entire calcium response kinetics in one experiment.

In normal FLIPR cell-based calcium mobilisation assays, membrane-permeable dyes bind Ca2+ and make it fluoresce more after an agonist interaction. Unfortunately, such assays can be subject to interference from various sources, including fluorescent compounds, Ca2+ ionophores, and compounds that permeabilize cell membranes; such nuisance compounds may obscure an agonist response and make allosteric modulator detection harder.

Molecular Devices has developed an innovative quenching dye to minimise interference from such substances. This novel dye offers superior light extinction in extracellular solutions and a wider assay window for screening quench-sensitive targets and receptors with low expression levels. Furthermore, the dye is resistant to anion exchange proteins such as organic anion transporters; hence, it requires minimal or no probenecid when used when analysing cell lines expressing such proteins.

Conventional GPCR FLIPR assays assess agonist activity using a two-step process; first, a known agonist is used to generate baseline responses before adding an unknown compound and measuring any changes in response from that change in response. This protocol significantly reduces screening costs by using fewer plates, cells, and reagents, while also decreasing robotic run time. Unfortunately, this approach requires off-line preparation of intermediate dilution plates, delaying screening time while increasing consumable costs.