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| More Content from High-Content Screening: Analyzing Spheroids in 3D When developing a HCS assay a critical aspect is the choice of cellular model. Multicellular “oids” (e.g. spheroids, organoids) have the potential to better predict the effects of drug candidates in vivo. However, 3D models tend to make all steps of the workflow more complex compared to 2D cultures. Generating large numbers of uniform spheroids at high quality for screening is one of the challenges. Using spheroids and cysts as examples, we show how to grow these models effectively using ULA coated U-bottom plates or low concentration gels. Dedicated tools like the Harmony® 4.8 high-content analysis software and PreciScan can also help users to explore 3D data sets and quantify in 3D changes such as volume, shape and number of nuclei per spheroid to better understand drug effects.
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| 3D Live Cell Organoid HCS Assay Development & Validation Epithelial-mesenchymal transition (EMT) is linked to the pathology of cancer with overwhelming evidence from literature associating EMT as a driving force for tumor progression and metastasis. Here, we describe the assay development and validation of a live-cell 3D multicellular tumor spheroid model for high-content screening (HCS) using a dual reporter EMT biosensor probe. The outcome of this dual reporter expression is engineered to identify compounds that inhibit, modulate, or reverse EMT in real-time.
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| Bioluminescence Resonance Energy Transfer for High-Content Imaging Fundamental processes in living cells are controlled by proteins, often acting through protein-protein interactions (PPIs), with other protein partners. Alterations in PPIs are linked to many diseases and are of increasing interest as potential drug targets. However, analyzing PPIs is challenging. One method that allows PPIs to be quantified is Bioluminescence Resonance Energy Transfer (BRET), which, in contrast to Förster Resonance Energy Transfer (FRET), does not require donor photo-excitation, circumventing autofluorescence and phototoxicity issues. Here, we present a high-content imaging application of BRET experiments using the Operetta CLS™ high-content analysis system and Harmony™ high-content imaging and analysis software.
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| Phenotypic Profiling of Autophagy One of the most important processes involved in maintaining homeostasis is autophagy, a process of degrading cellular components such as lipids, large protein complexes, or even whole organelles. Altered autophagy is found in various pathological conditions such as neurological diseases, cancer, and viral and bacterial infections. Here, we describe a high-content screening assay for autophagy, where different cancer cell lines were imaged using the Opera Phenix™ High Content Screening System and analyzed using the Harmony® High-Content Imaging and Analysis Software. In particular, we used PerkinElmer’s PhenoLOGIC™ machine learning. The building block-based analysis sequence consists of detecting spots in combination with calculating advanced morphological parameters, using them in the subsequent machine learning approach to distinguish between autophagy positive and negative cells.
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| Here, we describe the use of 3D human liver microtissues, coupled with the Opera Phenix™ High-Content Screening System, for assessing hepatobiliary transporter function in a complex 3D organotypic in vitro liver model system. Confocal imaging was used to visualize fine structures, and thus to assess the bile salt export pump (BSEP)-mediated efflux of cholyl-lysyl-fluorescein (CLF) and the multidrug resistance-associated protein 2 (MRP2)-mediated efflux of 5-chloromethylfluorescein diacetate (CMFDA) into the bile canaliculi.
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| Distinguishing Cell Lines by Phenotypic Profiling of the Nucleus Although the intention of high content screening is to extract as much relevant information as possible, a large percentage of high content screening assays only analyze a small number of image based properties. Here we show how nuclear staining can be used to differentiate cell lines from each other without any further staining or phenotypic markers, leveraging the high quality images derived from either the Opera Phenix™ or Operetta CLS™ systems in combination with the advanced texture and morphology tools of Harmony® High Content Imaging and Analysis Software.
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| FRET-based imaging assays are widely utilized to study protein-protein interactions or signal transduction processes in living cells. Here, we present a high content imaging assay to study the interaction between the pro-apoptotic protein Bad and the anti-apoptotic protein Bcl-XL in living MCF7 cells.
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| We present a High Content Screening application to analyze the integrity of the podocyte actin cytoskeleton. Using the Operetta® High Content Imaging System we acquired images of podocytes grown on CYTOO chips™ and analyzed drug-induced changes in podocyte morphology using the Harmony® High Content Imaging and Analysis Software.
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| Screening HDAC Inhibitors – A Workflow Comprising High Throughput and High Content Screening Here, we show how a combination of a biochemical assay, using AlphaLISA® technology, and a cell based high content assay with the Operetta® High Content Imaging System, can be used for identification of HDAC inhibitors. The approach presented demonstrates the potential power of combinatorial screening campaigns using the advantages of both target-based and phenotypic approaches.
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| Pushing the Boundaries of 3D Microtissue Analysis Using High Content Imaging We show how the boundaries of microtissue imaging can be pushed further by using the Opera® High Content Screening System (equipped with a water objective lens) in combination with a microtissue pre-treatment. The Scale aqueous reagent renders the biological sample optically more transparent and allows a greater imaging depth.
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| 3D cell culture methods are widely accepted as being more physiologically relevant than conventional 2D cell culture methods and are believed to improve the prediction of drug candidates at an early stage in the drug development process. In this study we describe the analysis of a spherical colon cancer microtissue model using the Operetta® High Content Imaging System.
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| Here, we present a high content analysis (HCA) based assay to study NF-kB signaling in living cells using the Operetta® High Content Imaging System and the Promega HaloTag® technology. By analyzing the acquired Operetta images with our Acapella® High Content Imaging and Analysis Software, we were able to track individual cells over time.
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| We utilized the HCA ImagAmpTM reagent kit in: (1) two typical high content analysis (HCA) assays for cytotoxicity, and in (2) a specific epigenetic assay monitoring the modulation of dimethylated lysine 9 residue on histone H3 (H3K9me2). Signal amplification with HCA ImagAmp is achieved through enzyme-mediated deposition of multiple fluorophores in close proximity to a given antigen.
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| Automation of Both Sample Preparation and Analysis of a Cell Migration Assay Here we demonstrate a successful automation of the sample preparation of Platypus Technologies’ Oris™ Pro Cell Migration Assay on a PerkinElmer JANUS® Automated Workstation. Analysis of cell migration using the Operetta® High Content Imaging System and the EnSpire® Multimode Plate Reader shows the assay to be a robust and reproducible quantification of cell motility.
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| Reagent-based Signal Amplification for High Content Analysis of PKCαLocalization in HeLaCells Here we demonstrate use of TSA for quantification of PKCαin unstimulatedand stimulated HeLacells using the Operetta® High Content Imaging System.
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| To investigate drug-induced cytotoxicity we used cryopreserved single-donor hepatocytes seeded into microplates in a conventional monolayer and treated them with three model hepatotoxins. After applying a convenient no-wash staining protocol live cells were imaged directly on the Operetta® High Content Screening System.
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| Chromobody® Based High Content Cell Cycle Analysis in Live Cells In this study we describe how single live cells can be followed through the entire cell cycle using a specific cell cycle protein binding cameloid antibody (Chromobody®) tagged with GFP (green fluorescent protein).
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| Measuring of Migration in a Fully Automated Imaging Based Approach Cell migration plays a major role in a variety of physiological events, such as immune response and wound healing. It also contributes to pathological processes such as metastasis. Understanding the molecular components of migration is crucial for discovering new targets to develop drugs that affect migration.
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| Previous research in basic and clinical neurosciences has improved the understanding of genetic disturbances associated with human neurodegenerative disease, which has permitted the generation of an increasing number of transgenic mouse models of Alzheimers disease, Parkinson disease and amyotrophic lateral sclerosis (ALS). Although the stru ctural and functional analysis of existing models has generated important insights, the detailed investigation of pathophysiological signal pathways in each mouse model remains a time – consuming challenge.
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| Image-based Quantification of HCS Cell-based Assays Using the Operetta and Harmony Imaging Platform The characterization of agents that inhibit cell proliferation and division is particularly important for drug discovery research. Both events can be analyzed using HCS approaches by multiplexing cell cycle-specific cellular targets. One technique is EdU staining, which detects the S-phase of the cell cycle through incorporation of the nucleoside analog Uridine into newly synthesized DNA strands. Furthermore, there are well validated protein markers available that are associated with certain cell cycle phases.
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| Cytotoxicity is a very complex process affecting multiple pathways and is not manifested by determining one morphological parameter. However, the ability to measure early indicators of toxicity is an essential part of drug discovery. Cell-based High Content Applications are a powerful tool for determining several events in cytotoxicity simultaneously. We describe a rapid and flexible dye-based high content cytotoxicity assay performed with the widely-used HepG2 (human hepatocellular carcinoma) cells.
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| One of the most important tasks in anticancer treatment is the inhibition of cell proliferation by interruption of the cell cycle. As the cell cycle is subdivided into the four phases G1-, S-, G2- and M-phase, there are diverse targets for arresting cells either during DNA replication in S-phase or during division in mitosis. Several cytoplasmic proteins like the cyclines have been identified to be proprietary in cell cycle control whereas Cyclin B1 in particular is essential for initiation of mitosis.
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| Apoptosis – the genetically coded program leading to the self-destruction of a cell – can be induced via two main pathways, the death receptor-mediated pathway, and the mitochondrial pathway. Induction of either finally results in the activation of caspases, a class of intracellular cytokine proteases which are considered to be the central components of the apoptotic response. By breaking down key cellular components that are required for maintaining normal cellular functions caspases are responsible for executing morphological and biochemical consequences directly or indirectly attributed to apoptosis.
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| G-protein coupled receptors (GPCRs) are an attractive drug target for several clinically relevant disorders. This is primarily due to their membrane location, ubiquitous expression and critical involvement in many mammalian physiological systems. While several assays targeted at downstream GPCR signalling events have allowed characterization of this receptor superfamily, there are few non-invasive techniques that allow analysis of the receptor-ligand interaction at the cellular level. |
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