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About Molecular Devices > Events > 2006 FLIPR User Forum

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2006 FLIPR User Forum

Thanks to everyone who attended our FLIPR User Forum in Seattle. We also thank all of our presenters, and especially our two session chairs, Charles Cheng of Neurogen Corporation, and David Harden of Bristol-Myers Squibb for all of their efforts to make this event a success.

The presentation topics are listed below and most are available for download in PDF format. If a presentation is not available here, please contact the author directly.

Use of Human Primary Cells in High Throughput Screening: A Reality in the Making using the FLIPR^TETRA in 1536-well format
Gary Allenby, Ph.D., AstraZeneca Pharmaceuticals
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The holy grail of high throughput screening (HTS) is to evaluate compounds in an as physiologically relevant environment as possible. Recent advances in liquid handling and reader technology have brought us one step closer to this reality. By using the FLIPR Tetra reader (Molecular Devices Corporation -MDC) in combination with the Echo 550 non-contact dispenser (Labcyte) we have performed GPCR-induced calcium mobilization assays in a cell line or human neutrophils in 1536 well format.

In detail: Our GPCR of interest is endogenously expressed on human neutrophils and was also stably transfected into HEK-293 cells. We originally screened the clonal cells to identify receptor antagonists in a 384 well FMAT whole cell-binding assay. We used the confirmed actives from this screen, together with standard compounds, to evaluate antagonism of GPCR-induced calcium mobilization in human neutrophils in 1536 well format using FLIPR^TETRA.

For spot testing, compounds were dispensed in triplicate using the Echo 550. Dilution curves for standard compounds were generated either by our standard dilution plate method or by the addition of differing volumes of stock material directly into assay plates using the versatility of the Echo 550 dispenser. Cells were dispensed using the DW-4 (MDC). Our data demonstrated good correlation between replicates in 1536 well format with excellent Z factors. Assays were relatively simple to establish and screen rapidly with good reagent cost savings.

The use of human primary neutrophils allowed compound screening in a native cell background and eliminated the slow step of cloning, expression and cell line generation, so speeding up the pre-clinical aspect of the drug-discovery process. Importantly, we have demonstrated the feasibility of human cell-based high throughput screening by using the FLIPR^TETRA reader in conjunction with the Echo 550 non-contact liquid dispenser.
The Advantages of Pin Tool Technology in the FLIPR^TETRA
Mitchell V. Hull, Genomics Institute of the Novartis Research Foundation

Pin tool technology can increase throughput and lower expenses while retaining an assay’s high quality. Disposable plastic tip boxes or 1536-tip gaskets can be costly and replacement steps can be difficult to automate and may require manual operation. Because pin tools are cleaned and reused during operation, no replacement steps are required. Moreover, since the FLIPR Tetra washes are performed simultaneously with plate reading, virtually no addition time is required to clean the pins. Another advantage is the pin tool’s ability to dispense low-volumes with high accuracy and precision, which eliminates the requirement of intermediate compound plates. Eliminating this step not only further cuts costs and increases speed, but also reduces the risks of compound precipitation that can be problematic in aqueous intermediate plates. However, this technology can present its own challenges. The advantages and caveats of pin tool technology along with strategies to ensure high quality assays will be addressed in this discussion.
FLIPR-based assays in Wyeth Screening Sciences
Veronica Soloveva, Ph.D., Wyeth Research

Cell based assays for identification of biologically active small molecules from chemical libraries are becoming increasingly popular for high throughput screening (HTS) aimed at a variety of drug targets. Three major components make the FLIPR-based assays successful are: good cells, optimal detection system and coordinated handling of the assay plates during experiments. The quality and scale of cell preparation have become critical and limiting factors in the process of screening. Diversity in cell culture technologies and methods of preparation enables the use of diverse cell types in screening campaigns. The TAP SelecT is an automated robotic system that can plate 100-300 plates of cells per day with inhuman accuracy and precision. In addition to plating cells, SelecT can also pass and expand cell lines. Some of the most fragile assays require good coordination between several independent processes during the run. This presentation will describe our custom designed, fully automated Thermo LAS robotic system with integrated FLIPR^TETRA and several additional peripheral elements such as a BioTek ELX washer unit, a PE Evolution pipettor and PE FlexDrop dispenser. Finally, good variety of different Ca-indicator assay kits is available for comparison studies to achieve best results with weakly responding Gq coupled receptors. We will discuss our strategies of assay optimization during the assay development phase before each screening campaign.
Multiplexed GPCR Agonist Assay in the 384-well FLIPR system: Screening and Hit De-convolution
Charles S. Cheng, M.S., Neurogen Corporation

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Methods and strategies for compound screening which offer improved throughput while containing costs is a goal of drug discovery efforts. Using the 384 well FLIPR platform an assay format was developed for simultaneously screening of multiple GPCR targets in agonist mode. Although the basic technique being employed, co-seeding of multiple cell-lines into the same wells of the assay plate, has been practiced elsewhere newly employed logistical solutions for executing a multiplexed assay yielded considerable benefits in assay throughput vs. a mono-cell line screening approach and simplified data de-convolution. The particular screening effort being described examined three target receptors simultaneously. A proprietary biosensor was utilized in two of the three target receptor cell-lines which allowed two of the target receptors to be of the Gs type while the third target receptor was of the Gq type. Development work was needed to assure signal strength compatibility for all the cell-lines involved particularly because there were different signaling pathways being utilized by the different target receptors. The information generated by this multiplexed screening and de-convolution approach was successfully used for a simple and fast identification of compounds with target agonist activity. Multiple compounds with receptor specific agonist activity were identified at one of three target receptors in our assay, melanocortin 1 receptor. These compounds had potency values over a broad range up to approximately 100 nM. Overall, this screening and data de-convolution format was used to screen a specific compound set for agonist activity at three different GPCR targets for the cost and time expenditure that previously would have been need to screen at only one target.
New users assay development experiences using Transfluor
Myles Fennell, Ph.D., Wyeth

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Agonist dependent internalization of GPCR’s, a common property of these receptors contributing to the desensitization of agonist-mediated responses, can be used as a measure of functional receptor activation. We have developed a series of assays using the Transfluor GPCR activation system, which utilizes GFP-coupled b-arrestin-2 re-distribution as an index of receptor activation and internalization. Translocation of b-arrestin from a uniform to punctuate distribution following agonist treatment was measured and quantified using the Spot Detector algorithm on a Cellomics Arrayscan. As a proof of concept and validation study we overexpressed two RNA edited variants of the 5HT2C receptor (INI and VNI), with different affinities and functional responses to serototin. The methods used for transient receptor overexpression were baculovirus and the Amaxa nucleoporator system, both of which are more efficient than lipid based transfection methods. In addition to determining the pharmacology of agonist-induced receptor internalization we optimized agonist-independent receptor activation by expressing constitutively active G-protein coupled receptor kinase (GRK-Lite), which phosphorylates the C-terminal tail of GPCR’s following their activation and leads to recruitment of b-arrestin and receptor internalization. As well as developing assays for 5HT2C receptor activation and pharmacology with Transfluor, we also compare and contrast with functional data using FLIPR measurements and receptor internalization using EGFP-coupled 5HT2c receptors.
The ImageXpressULTRA™: The Benchtop, High-throughput, Point-scanning Confocal Imaging Syste
Sylvia de Bruin, Molecular Devices Corporation

Molecular Devices (MDC) has a complete toolset for High Content Screening (HCS), consisting of two fully-integrated high throughput imaging systems and a suite of imaging software for image acquisition, automated analysis and cellular informatics. The focal point of MDC’s HCS solution is a new generation of automated microscopes: the widefield ImageXpressMICRO and the confocal ImageXpressULTRA. These instruments, developed to address the limitations of older generation systems, are cost-effective bench-top systems combining state-of-the-art engineering with industry-leading imaging software. Both systems share high-speed laser auto-focus for increased throughput and better than 100 nm resolution for sample positioning and focus. ImageXpressULTRA is a modular, true point-scanning confocal imager utilizing solid-state lasers, photomultiplier tubes (PMTs), and self-aligning optics, providing simultaneous multi-channel acquisition for up to four different wavelengths. The ImageXpressULTRA has a fully configurable pinhole for adjustment of image size and scan length. Modules include choice of objectives, emission filters and robotic microplate handling.
MetaXpress – Making Image Analysis Simple
Anna Hastings, Molecular Devices Corporation

Molecular Devices (MDC) has a complete toolset for High Content Screening (HCS), consisting of two fully-integrated high throughput imaging systems and a suite of imaging software for image acquisition, automated analysis and cellular informatics. As part of this toolset, the MetaXpress™ software package provides robust, automated, and easy-to-use image analysis tools, enabling researchers to get the most out of their MDC HCS instruments. Built on the industry-standard MetaMorph imaging platform, MetaXpress streamlines HCS analysis for a wide variety of assays. Simple, intuitive, and modular set-up yields robust results immediately, while utilizing sophisticated state-of-the-art analysis algorithms within. Separate biology-specific modules are available for an extensive and continually growing list of assays, including GPCR internalization, Cell Health, Nuclear Translocation and Neurite Outgrowth. Specially tuned and optimized High Throughput (HT) modules simplify the analysis of large compound libraries. In this session, we will show how to easily configure MetaXpress modules, demonstrate HT analysis of a Transfluor® microplate, and review results.

AcuityXpress™: New Developments in Cellular Informatics
Pierre Turpin, Ph.D., Molecular Devices Corporation

An Evaluation of No Wash dyes for FLIPR Calcium Mobilization Assays
Donna Terenzio, M.S., Boehringer-Ingelheim Pharmaceuticals, Inc.

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In HTS, the utilization of cell-based calcium functional assays, such as those run on FLIPR (Molecular Devices Corp.), have increased dramatically over the past several years. These assays play an integral role in preliminary compound assessment by providing both binding and functional details of the receptor/compound interaction. Historically, calcium binding sensing dyes used in these assays, such as fluo-3 and fluo-4, required the removal of unincorporated, extracellular dye before a response could be measured. This wash step resulted in greater variability in assay performance with respect to S:B, Z’ and signal amplitude. The new generation of calcium binding sensing dyes are “no-wash” dyes, which obviate the removal of unincorporated dye by the addition of quenchers to the dye solutions. Molecular Devices new Calcium 4 no-wash dye kit is among these. This dye is evaluated for all aspects of performance compared to other commercially available dyes. As well compound activity can be misinterpreted by compound autofluorescence, thus a preliminary evaluation of a new red-shifted dye is also discussed.

Evaluation of FLIPR^TETRA to support Aequorin Assays
Paru Rao, Glaxo SmithKline

Aequorin is a luminescent photoprotein derived from a jelly fish Aequoria victoria that in presence of calcium, undergoes conformational change and emits light and has been used as intracellular calcium indicator which is typically quantitated on a luminometer. FLIPR has been extensively used for a decade to identify agonists and antagonists for GPCRs and ion channels by monitoring intracellular calcium mobilization using fluorescent dyes such as Fluo-4 or Fluo-3. Here we present data to show that FLIPR^TETRA not only can be used for fluorescence based assays but also for luminescence based assays.

Application of Photina™ for Luminescence-based Detection of Ca2+ Signaling on FLIPR^TETRA
Sabrina Corazza, Ph.D., Axxam s.r.l

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Ca2+ activated photoproteins are important tools for analysing all aspects of Ca2+ mediated signal transduction processes in mammalian cells. One of their characteristics is the immediate photon release (flash luminescence) upon Ca2+ binding to the coelenterazine-photoprotein complex, which makes this system extremely useful for studying rapid receptor-ligand interactions or fast acting ion channels if Ca2+ mobilization is involved.

Photina™ is an extremely bright photoprotein generated at Axxam and optimized for the use in HTS campaigns. Its character allows the detection of rapid increases in intracellular Ca2+ concentration and it is featured by the absence of any background activity with a consequent high signal-to-noise ratio and a broad range of detection sensitivity.

The presentation will show data obtained with Photina™ cell lines on the FLIPR^TETRA. All the Photina™ cell lines used in this study are containing the mitochondrial version of Photina™ (mito-Photina™) that is particularly suited for studying Gaq mediated activation. Data obtained show that it is possible to detect Photina™ luminescence on FLIPR^TETRA with a very good reproducibility. The obtained EC50 and IC50 values are always in good agreement with the literature data. Moreover, since the flash luminescence signal generated by Photina™ cell lines is very robust, this photoprotein was also tested in the 1536 MTP format. The data confirmed, that Photina™ is well suited also for the 1536 well applications on the FLIPR^TETRA.

In addition, the same Photina™ expressing cell lines used for luminescence detection have been run in parallel using the MDC Calcium and Calcium 3 fluorescent kits. The EC50 and IC50 values obtained using Photina™ as read out are similar to the one obtained using the fluorescent dyes. Anyhow the great advantage of using Photina™ resides on the fact that the basal value of Photina™ is very low compared to fluorescent dyes, allowing a higher dynamic range and an higher signal to background value. First experiments were also carried out testing the performance of Photina™ on FLIPR^TETRA with a suspension protocol. Data obtained demonstrate that Photina™ flash luminescence can be detected on FLIPR^TETRA using the cell-suspension approach.

User of FLIPR-based detection of thallium influx in high-throughput screening for potassium channel modulators
David Harden, MS, MBA, Bristol-Myers Squibb

Potassium (K+) channels are emerging as important therapeutic targets as well as drug safety liabilities. Although several screening approaches exist, a novel thallium (Tl+) flux assay was developed at Bristol-Myers Squibb (BMS) that provides a direct, robust, and high-throughput measurement of K+ channel activity using standard screening equipment. We have applied this technique to voltage-gated and ligand-gated K+ channels to identify novel activators and inhibitors from within the entire BMS screening collection. Potency and efficacy values are readily obtained using the Tl+ flux assay and these values correlate well with corresponding values obtained using whole cell patch clamp recording for both channel activators and inhibitors within and across chemotypes. Representative results will be presented for voltage-gated and ligand-gated channel modulators. These data illustrate the utility of this technique in lead identification as well as lead optimization.

Characterization of biogenic amine transport using fluorescent substrates with the FlexStation
John N. Mason, Ph.D., Vanderbilt University Medical Center

Neurotransmitters transporters located on presynaptic membranes of neurons terminate neurotransmission efficient, transporter-mediated clearance. The norepinephrine, dopamine and serotonin transporters (NET, DAT, and SERT) are homologous transporters that exhibit overlapping substrate and antagonist recognition. Transporter activity is predominately measured by the ability of cultured cells or synaptosomes to accumulate tritiated substrates. Several limitations of this method include low time and spatial resolution, the inability to acquire repeated analyses on single samples, and the expense and biohazard of radioisotopes. Therefore, we have developed methods that monitor the accumulation of fluorescent substrates using confocal microscopy for single cell resolution and automated fluorimeters (e.g. FlexStation, Molecular Devices) for high-throughput applications. Our lab has investigated two fluorescent molecules (ASP+ and IDT307) as substrates for biogenic amine transporters. Once taken into the cell and excited, each of these molecules demonstrate environmentally sensitive fluorescence, enabling their accumulation to be monitored following UV excitation. ASP+ (4-(4-dimethylaminostyryl)-N-methylpyridinium) is a fluorescent analog of MPP+, which has specificity for the NET and DAT as well as organic cation transporters, recently characterized by our lab and others (Mehrens et al. 2000; Schwartz et al. 2003, 2005; Mason et al., 2005). Using confocal techniques, we have demonstrated that ASP+ accumulation can reveal sites of transport at the level of single neuronal varicosities. To overcome limitations with ASP+ in detecting specific uptake for SERT, we have synthesized and characterized the uptake of a novel compound, IDT307. Using both confocal microscopy and the FlexStation with stably transfected mammalian cells, we have demonstrated that IDT307 permits real-time monitoring of SERT and NET suitable for screens targeting the development of regulators and antagonists of these transporters. Given the large number of transporter genes and our success monitoring biogenic amine transport, fluorescence-based approaches would appear to have wide utility for the analysis of transport proteins.

By using the automated FlexStation™ to monitor the accumulation we have demonstrated the utility of this approach for characterized transporter function, to screen inhibitor and substrate potencies with in real-time using a nonradioactive substrate, and to characterize transporter expression in native tissues.

Of the 1200 identified transporters the 90% lack assays for measuring their function. The fluorescent substrate assay is an approach that is inexpensive, non-toxic. Its ability to provide repeated measurements enables monitoring of real time effects. We are presently designing additional compounds with greater specificity. The use of substrates with different wavelength offers the ability to multiplex.

Posters

Evaluation of Ion Channels Activity Using the FLIPR System
Dr. Sabrina Corazza, Axxam srl

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A fluorescence-based FLIPR® assay for potassium channels using a thallium sensitive probe and proprietary masking dye technology
Jeff Quast, Rheka Thomas, Sukanta Bhattacharyya, Annegret Boge and Rich Sportsman

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Time-resolved Fluorescence Resonance Energy Transfer detection for Phosphodiesterases using the non-radioactive and antibody independent IMAP® platform
Francisco Ramirez, Rich Sportsman and Annegret Boge

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Time-resolved Fluorescence Resonance Energy Transfer detection for Lipid kinases using the IMAP® platform
Joyce Itatani, Rich Sportsman and Annegret Boge
Molecular Devices Corporation

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Comparison of calcium flux assays across multiple GPCRs
Annegret Boge, Richard Sportsman (Molecular Devices Corp.)
Helena Mancebo, Jeng-Horng Her, Jianfu Jeffrey Wang (Multispan, Inc.)

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	Event Presentations 2008 Automated Ephys Users Meeting 2007 Axon GenePix Web Seminar Series 2007 Imaging Users Meeting 2007 Arcturus LCM Users Meeting 2007 FLIPR Drug Discovery Forum 2007 Automated Ephys Users Meeting 2006 Automated Ephys Users Meeting 2006 FLIPR User Meeting Presentations 2005 MetaMorph Users Meeting 2005 Axon Cellular Neurosciences Users Meeting 2005 Automated Ephys Users Meeting 2004 Drug Discovery Conference 2003 Drug Discovery Conference 2003 SBS Conference 2003 European Users Meeting 2002 Drug Discovery Conference 2001 Drug Discovery Conference 2000 Drug Discovery Conference