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Operetta Application Notes
Liver toxicity remains one of the main reasons for drug failure in clinical trials. Improving preclinical toxicity testing is a major prerequisite for improving the drug de-risking process. Early compound de-risking decisions require in vitro data with the highest possible biological relevance. Three dimensional (3D) microtissues, or spheroids, are one of the most well characterized models for 3D culture and cell-based drug screening due to their reproducibility and similarity to the in vivo situation.
Cell migration is an essential component of metastatic dissemination of tumor cells from the primary tumor to local and distant sites. For many types of cancer, cancer cells respond to stimulation with growth factors or chemokines with an increased migration – a process called chemokinesis. Consequently, pathways which render tumor cells susceptible to chemokinesis are interesting drug targets for reducing the risk of metastasis in cancer patients (Roussos et al., 2011).
In vivo cells form three-dimensional (3D) microenvironments through cell-cell and cell-matrix interactions to create complex tissues and perform higher level functions. Biochemical signals and nutrient gradients are essential for tissue functioning and therefore also physiological cell behavior. Conventional two-dimensional cell culture does not provide such a complex microenvironment and consequently cells can lose some of their physiological properties (Pamploni et al., 2007).
To allow efficient screening of large compound libraries, intelligent screening workflows are needed that combine different assay formats and multiplex various readouts, including phenotypic information, to provide a more holistic view of compound effects. This application note, developed in collaboration with the European ScreeningPort, describes a workflow for HDAC inhibitor screening and demonstrates the combination of a high throughput AlphaLISA® assay and a high content assay using the Operetta® High Content Imaging System.
Drug-induced nephrotoxicity is one of the most common reasons for the failure of new chemical entities in clinical trials during drug development. It is also a serious problem for approved drugs such as contrast media [Deray, 2006] or the widely-used non-steroidal anti-inflammatory drugs (NSAIDs) [Clive and Stoff, 1984]. Here we present a High Content Screening application in collaboration with Evotec AG, to analyze the integrity of the podocyte actin cytoskeleton.
Here we show how the Opera® High Content Screening System and Operetta® High Content Imaging System can be used with different software products in the PerkinElmer portfolio to provide an in-depth analysis of screening data. Using different application examples, we show how the Volocity® 3D Image Analysis Software can be used for advanced 3D visualization and single cell tracking and how the Columbus™ Image Data Storage and Analysis System can be used for secondary analysis of screening data.
Cathepsins are proteases, detected at basal levels in all living cells (Turk et. al., 2001), that are known to play an important role in several physiological processes such as lysosomal protein degradation, antigen presentation and apoptosis. However, cathepsins are also associated with pathological conditions and diseases such as cancer, osteoporosis and bronchial asthma (Turk et. al., 2001; Zavasnik-Bergant & Turk, 2006; Vasiljeva et. al., 2007).
The NF-κB family of transcription factors was identified more than 20 years ago and today it is well established that NF-κB plays a pivotal role in all aspects of immunity and cellular response to pathogens [Hayden and Ghosh, 2008]. Aberrant activation of NF-κB signaling is associated with inflammatory diseases such as rheumatoid arthritis (Criswell, 2010) and asthma (Broide et al., 2005).
Human mesenchymal stem cells (hMSCs) are multipotent cells present in the bone marrow that can replicate as undifferentiated cells. They have the potential to differentiate to lineages of mesenchymal tissues such as bone, cartilage, muscle and fat. When cultured in vitro, these cells can undergo osteogenic, chondrogenic or adipogenic differentiation depending on the culture conditions (Pittenger et al., 1999).
Embryoid bodies (EBs) are spherical three-dimensional cell aggregates derived from embryonic stem cells (ESCs) which serve as model systems for embryonic development. The formation of EBs allows the study of cellular and molecular interactions in a three-dimensional manner in the early stages of embryogenesis.
HCA ImagAmp™ is an amplification system designed to provide significant enhancement of target-specific fluorescence signals and to improve sensitivity in immunofluorescencebased assays. Amplification is achieved through enzyme-mediated deposition of multiple fluorophores in close proximity to a given target.
The use of cultured primary human hepatocytes in predictive in vitro studies of drug metabolism and hepatotoxicity, in different stages of the drug discovery process, has increased in recent years. Primary liver cells are recognized to be the closest and most relevant in vitro model to the human liver as they express the entire hepatic contingent of drug-metabolizing enzymes and transporter proteins.
Cell invasion is of major importance in many biological processes such as embryonic development, inflammatory responses, the repair of injured tissues, and tumor formation and metastasis [Horwitz and Webb, 2003]. During metastasis, individual cancer cells leave the initial tumor mass, enter the circulatory system and move into distinct parts of the body (Lauffenburger and Horwitz, 1996).
Protein kinase C alpha (PKCα) is associated with a wide variety of cellular processes including proliferation, adhesion and motility. Over expression of PKCα appears to be associated with certain forms of cancer, and it has been identified as a biomarker for poor prognosis in breast cancer ([Lønne et al., 2010). Upon activation with phorbol esters, PKCα translocates from the cytosol to the plasma membrane [Chun et al., 1996]. For this reason, it is best studied with a high content analysis method that provides quantitative data on subcellular localization.
Here, we present a different approach to the analysis of plasma membrane bound PKCa signal which is independent of any membrane dye or spot accumulation. Our plasma membrane texture-based approach has been shown to be very robust, and accurately quantifies PKCa located in the ridge-like structures of membrane between neighboring cells.
In recent studies it was suggested that a more soluble form of Htt represents the toxic species of Htt protein [Arrasate et al., 2004] rather than the aggregate form. An automated measurement and analysis of Htt protein in ST Hdh Q111/111 cells offers a promising basis for the development of new therapeutics against the disease.
Here, we present a high throughput High Content Analysis (HCA) assay for the quantification of the specific, more soluble conformation of the Htt protein which is located in the perinuclear region.
Cell migration plays a major role in a variety of physiological events, such as immune response and wound healing. In addition, cell migration can also contribute to pathological processes including metastasis [Lauffenburger et al., 1996; Horwitz et al., 2003]. Understanding the molecular components of migration is crucial for discovering new targets to develop drugs that affect migration. Identification of new therapeutics that manipulate cell migration would benefit from recent advancements in High Content Screening (HCS) technology.
Here, we present a method to analyze cell migration using the Operetta / Harmony HCS platform and the Oris(tm) Cell Migration Assay from Platypus Technologies, LLC.
Apoptosis, the genetically encoded program that leads to the self destruction of a cell, can be induced via two main pathways: the death receptor-mediated pathway, and the mitochondrial pathway [Hengartner MO, 2000]. Induction of either will finally result in the activation of caspases, a class of intracellular cytokine proteases which are considered to be the central components of the apoptotic response.
Cytotoxicity is a very complex process affecting multiple pathways. The ability to measure early indicators of toxicity is an essential part of drug discovery. An important approach to the detection of compound toxicity is a multiparametric analysis at the level of individual cells using High Content Analysis (HCA) applications [Abraham et al., 2008]. Such cell-based assays, which assess several cytotoxic events in a variety of relevant pathways.
The cell cycle is a ubiquitous, complex process that underlines the growth and proliferation of cells, organismal development, regulation of DNA damage repair and tissue hyperplasia as a response to injury and diseases such as cancer. The characterization of pharmacological compounds that regulate cell proliferation and division is therefore particularly important for drug discovery research.