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Development of HTS of Primary Lung Epithelial Cells for Target Discovery in Pulmonary Fibrosis
Edo Elstak, Senior Scientist, Galapagos BV
Fibrosis observed in (chronic) lung diseases is thought to be reflected, amongst others, by epithelial to mesenchymal transition (EMT). Primary human bronchial epithelial cells (HBEC) are therefore an excellent starting point for assay development and target discovery in fibrosis. Galapagos has ample experience in assay development with their SilenceSelect® adenoviral shRNA technology and high content imaging. Epithelial cells isolated from primary human lung tissue were acquired and transduced with shRNA adenovirus during development to confluent monolayers. EMT was induced using a cocktail containing TGFß and the progress of EMT was quantified with the GE IN Cell 2000, using a segmentation protocol developed to detect E-cadherin and Fibronectin patterns. shRNA viruses that target known signaling pathways in EMT validated our approach and SilenceSelect® library screening may thus serve as a suitable tool to identify novel targets in excessive pulmonary fibrosis.
An In-vitro Model of Acute Epilepsy Suitable for MTS
Eva-Maria Tomic, , Hamamatsu Photonics Deutschland GmbH
Many broad spectrum antiepileptic drugs (AEDs) have been discovered following a phenotypic screening strategy. Here we describe a robust and reproducible Ca2+ oscillation assay in 384-well format. Synchronized Ca2+ oscillations induced by experimental conditions with 0.1 mM Mg2+
were measured in primary culture of embryonic hippocampal rat neurons. More than 40 references compounds have been subjected to this assay. Our results may reveal the molecular mechanisms mediating the effects of these compounds, such as signaling via ion channels/GPCRs, reuptake
systems, and enzymatic functions.
This assay is suitable for MTS and provides an effective way for identification of potential AEDs with new modes of action.
Instant Transient Transfection of Bulk Frozen Cells
Monique Oberlaender, , Cell Culture Service
Frozen Instant Cells are high-density stocks of cryopreserved cells which don’t need to be cultivated before they are used for a subsequent application, e.g. a cell based assay. After thawing these cells can be directly seeded into assay plates and immediately regain their full assay performance. By using assay-ready frozen cells the production of the cells can be timely and spatially separated from their actual use. This is of great advantage in particular for high-throughput screening (HTS) where a continuous and reliable supply of high quality cells is important and very often a bottle neck. For this reason the use of assay-ready frozen cells became a broadly accepted technology in cell based drug screening within the recent years.
The best practise of freezing cells is still to use small vials of 1 to 5 ml which can be stored at ultra-deep temperatures. For an HTS campaign however, the use of small aliquots sometimes becomes quite laborious. 20 to 50 cryovials have to be thawed, decapped and pooled in order to supply the cells for a single screening day. Assay-ready cells which are frozen in bulky aliquots would very much reduce this effort.
Not only for HTS campaigns but also for bulk transfections the use of large aliquots of ready-to-use cells would be helpful. Transient transfection of bulk cells has become a straight forward alternative to recombinant cell lines for cell based drug screening. While recently the restriction of scalability has been very well addressed by the Scalable Electroporation Device (STXä) from MaxCyte®, the supply of bulk cells for transfection is still a bottle neck.
We here demonstrate that assay-ready cells can be frozen in large volumes of up to 100 ml. Cells which had been frozen either in 50ml conical centrifugation tubes or ethylene vinyl acetate (EVA) freezing bags display a high viability and fitness after thawing. We also show that cells frozen in large containers can be directly used for transient bulk transfection.
High Throughput imaging of cell based assays and cellular models using microplate cytometry
Dawn Browning, Conference Organiser, TTP Labtech Ltd
Microscope-based, high-content instruments are used for many cell based assays in high content screening (HCS). For efficient and rapid resolution most assays require the use of higher resolutions which entail lengthy read times, using single colours and analyzing only a small percentage of the total number of cells in a well to keep plate read times at a minimum. Using Acumen eX3® with its wide field objective lens and “on-the-fly” laser scanning capabilities, it is possible to rapidly scan entire wells, obtaining statistically robust data from a number of assays relevant to the oncology research such as cell cycle analysis, cell migration, cell surface marker analysis and cell signalling.
In this study, we demonstrate the use of Acumen to assess the effect of inhibitory compounds on the mitotic index and cell cycle. In addition, by employing the multiplexing capabilities of Acumen eX3 cell enumeration was carried out by counterstaining with PI. Using a second fluorescently labelled antibody conjugate against the phosphorylated protein, phospho-histone H3, the effect of the inhibition of two kinases p38 and Chk 1 on G2 mediated checkpoint arrest was quantified.
With its high depth of field, we demonstrate that Acumen is capable of scanning larger organisms such as C.elegans and even Zebrafish to provide information on multi-cellular drug interactions at an early screening stage.
Built in software offers the flexibility of exporting whole well open source TIFF images for batch processing by third party image analysis software packages. This new screening paradigm represents a major breakthrough in how microplate cytometry can be applied to complex cellular models since By combining image-processing methodology with rapid cytometric it is now possible to develop whole organism or tissue based assays models for HCS.
Homogeneous Binding Assay for Hybridoma Screening: Comparison of Two Laser Scanning Systems
Dawn Browning, Conference Organiser, TTP Labtech Ltd
Monoclonal antibody therapy represents one of the most rapidly expanding and exciting segments of the pharmaceutical industry today. This has placed unprecedented demands on the antibody discovery process that has traditionally relied on assay formats such as ELISA and flow cytometry.
In this study, we compare a mirrorball â high sensitivity microplate cytometer (TTP LabTech) with the ABI 8200 system (Applied Biosystems , FMAT®) for a homogeneous (no wash) homogenous binding assay. mirrorball is a new bench top laser scanning system that rapidly and simultaneously acquires and processes data from the whole well to provide simple readouts for antibody binding.
The combination of mirrorball and no-wash assay protocols allows researchers to screen antibody supernatants with minimal reagent additions, no wash steps and achieve excellent sensitivity with only 5-10 microlitres of sample. Using the mirrorball’s unique ability to scan simultaneously with up to three lasers, it is also possible to multiplex antigens, for example target antigen and counter-screen.
In this poster, we demonstrate a direct comparison of these systems for a representative hybridoma screen.
HTRF Cellular Kinase Assays to Investigate Major Signaling Pathways
Frederique Hudon, HTRF Operational Marketing, Cisbio Bioassays
Due to their involvement in neurological, oncologic or inflammatory disorders, the G-Protein coupled receptors and the kinases families assemble almost all therapeutic targets. Therefore numerous current pharmaceutical programs aim to develop biomolecules; either biologics (e.g. therapeutic antibodies, peptidic or organic small molecules) or permeant small molecules (ATP-competitive in the case of the kinase family). A complete pharmacological characterization is therefore required for a drug’s comprehensive mechanism of action. During recent years, the majority of compounds of interest have been investigated through biochemical assays using purified recombinant enzymes to indicate the biological relevancy of this strategy. Recently the scientific community has adopted the analysis of signaling pathways in response to drug treatment, in a cellular culture model. Attentive to creating innovative, high-performance relevant products, Cisbio Bioassays has developed a panel of cell-based assays dedicated to studying the behavior of a compound toward a signaling pathway.
This poster illustrates the application of these HTRF® cellular kinase assays (EGFR, HER2, Erk, Akt, Stat3, p38 and IKKbeta) for the detection of endogenous cell surface receptor activation in various cell types. Using a common simple protocol, these assays are a rapid alternative to traditional methods such as the Western blot or ELISA.
HTRF cellular kinase assays to address ligand-gated channels: Example of the a7 nicotinic acetylcholine receptor
Frederique Hudon, HTRF Operational Marketing, Cisbio Bioassays
The a7 nicotinic acetylcholine receptor (a7 nAChR), is the most studied ion channel of the ligand-gated channel family (LGICs) which are implicated in several neuropsychiatric and neurodegenerative diseases. According to the literature, several kinase phosphorylation pathways are a7 nAChRmediated. This poster illustrates the applicability of measuring phospho-Erk to address a7 nAChR. Phosphorylated Erk1/2 measurement using the Cellul’erk kit was compared to calcium signaling (FlexStation) using several specific pharmacological compounds including PNU-120596 (positive allosteric modulator: PAM), PNU-282987 (agonist), and Methyllycaconitine and a-Bungarotoxin (antagonists). Cellul’erk is a ready-to-use cell-based assay based on HTRF technology. Using a simple protocol, this assay is a robust, reliable and rapid alternative to more conventional technologies such as electrophysiology, Western Blot, Calcium flux measurement or ELISA.
Automation of a Novel Cell-Based ELISA for Cell Signaling Pathway Analysis
Wendy Goodrich, Applications Scientist, BioTek Instruments Inc
Monitoring and quantifying cell signaling pathways is critical for understanding the behavior of cell processes and many disease states. Protein kinases involved in these cellular cascades play many diverse biological roles including normal growth and development; their aberrant behavior is linked to a number of infirm states including cancer. A novel ELISA assay has been developed for detection of cell based signal transduction events. In addition to offering simplicity, speed, and flexibility, the technology is a highly sensitive technique that lends itself to automation at multiple levels including seeding cells in microplates; cell media exchanges; serial dilution and transfer steps; assay reagent additions; microplate washing; and, signal detection. Assay performance studies were conducted using lysate kit controls to verify optimal automation performance for S/B, Z’, and intra- and inter-assay precision. A ‘one well one day’ cell based assay protocol was developed and used for pharmacology assessment for agonist/antagonist analysis of the PI-3/AKT/mTOR signaling pathway. Procedures demonstrated here are well suited for validating automation of model system protocol development.
Wide Field-of-View Microlens-Enhanced Dual Nipkow Disk Confocal Technology
David Lorenz, Associate Director, Wako Automation
High Content Analysis (HCA) requires high throughput to shorten the experiment period and increased accuracy through detailed analysis of large amounts of cell data. If priority is given to image-capturing speed and a low- power objective lens is used, images of a wide area can be taken, but resolution will be reduced, making it difficult to observe detailed phenomena in the cells. The use of an objective lens with high magnification and a narrow field of view to observe detailed cell conditions requires an enormous number of images and can be time consuming. The HCA requires both wide-view and high-resolution imaging.
This poster describes a novel wide field-of-view (FOV) microlens-enhanced Nipkow disk confocal technology that utilizes sCMOS camera, as the core technology of the High-Throughput Cytological Discovery System, CellVoyager CV7000.
A Miniaturized and Automated P450-Glo™ Screening System Using the Echo® Liquid Handler
Jesse Monsma, Marketing Coordinator, Labcyte
Early assessment of cytochrome P450 (CYP) inhibition has become an essential component of drug discovery screening. The CYP3A4 enzyme is the most abundant enzyme in the liver and plays a major role in metabolizing many xenobiotics in humans. The Promega P450-Glo™ CYP3A4 Assay (Luciferin-IPA) offers an extremely sensitive, high throughput and specific luminescence assay for the examination of CYP3A4 inhibition with pooled Human Liver Microsomes. The Echo 555 liquid handler provides precise and accurate acoustic transfer free of cross-contamination risk. Here, we demonstrate optimization of the P450-Glo™ Assay with Human Liver Microsomes (HLM) in low volume format using the Echo 555 liquid handler. We show that the Echo 555 liquid handler can transfer microsomes, inhibitors and substrate in nanoliter volumes, providing a sensitive and robust small scale assay. IC50 results for four known CYP3A4 inhibitors using our miniaturized assay compared favorably to previously reported values in the literature.
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