For Research Use Only. Not for Use in Diagnostic Procedures.
Opera Application Notes
Three-dimensional (3D) cell culture methods are widely accepted as being more physiologically relevant than conventional two-dimensional (2D) methods. Cellular functions and responses that exist in tissues are often lost in 2D cell cultures, limiting their predictive capability for drug efficacy screening [Pampaloni et al., 2007; Windus et al., 2012]. 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 tissues in an organism.
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.
Using the brightfield option of the Opera® High Content Screening System, a digital phase image can be constructed using two brightfield images acquired at different Z planes. The digital phase image generated has comparable properties to an image showing fluorescently labeled cells with high signal to noise ratios and allows image segmentation into single cells.
Drug compounds with genotoxic side effects give rise to micronuclei (MN) formation in cell cultures. As a result, in vitro MN assays have become an important tool for assessing and pre-screening the mutagenic potential of drug candidates in the early stages of the drug discovery process. A micronucleus is a small DNA fragment in the cytoplasm of interphase cells – an erratic, small, extra nucleus. Micronuclei contain fragments or whole chromosomes that were not incorporated into the daughter cell nuclei during mitosis [Norppa and Falck, 2003].
Chromobodies® are a new class of fluorescent antibodies which are characterized by their extremely small size, high stability and excellent reproducibility. Most importantly, Chromobodies can be used in live cells to target and trace their endogenous antigens through different subcellular compartments, which is not possible with conventional antibodies [Rothbauer et al., 2006]. Unlike classical GFP-fusion proteins, Chromobodies have the potential to detect and visualize non-protein components or specific post translational modifications of target proteins. These characteristics render them valuable tools as direct live cell biomarkers on High Content Screening platforms.
Transcription factors such as FKHR function as key regulators in e.g. insulin signalling, cell cycle progression and apoptosis downstream of phosphoinositide 3-kinase (PI3K). Inactive FKHR is cytoplasmic but is rapidly imported to the nucleus upon inactivation of the PI3K/Akt pathway. The Forkhead Redistribution® Assay is designed to assay for inducers of FKHR translocation in FKHRGFP fusion protein expressing cells. Monitoring the translocation of the fusion protein from the cytosol to the nucleus requires a high resolution imaging technique.
The regeneration of neurons represents a promising strategy for drugs targeted against neurodegenerative injuries and disorders such as Alzheimer’s and Parkinson’s disease [Geerts et al., 2005]. Therefore, the development of new therapies is focused on identifying molecules that affect the differentiation of neurons and neurite outgrowth. For this purpose automated measurement and analysis of neuronal cells is essential for neuroscience research and drug discovery.
Endothelins (ETs) are a class of peptide hormones with strong vasoactive properties [Bagnato & Natali, 2004]. ETs function by interacting with their G protein-coupled receptors (GPCRs). There are two known ET subtypes, ETA and ETB [Bremnes et al., 2000] The ETA receptor (ETAR) is localized on the plasma membrane until stimulated by endothelins such as endothelin-1 (ET-1), towards which ETAR shows the highest binding affinity.
The ability to measure early indicators of toxicity is an essential part of drug discovery. In vitro cytotoxicity assays involving tissue specific cell cultures are considered to be valuable predictors for human drug toxicity. The potential of such in vitro assays lies in the fact that several cytotoxic effects in a variety of relevant pathways can be assessed simultaneously.
Bruton’s tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase (PTK) crucial for B-cell development and differentiation. It binds to phosphatidylinositol-3,4,5- trísphosphate (PIP3) through the Btk pleckstrin homology (PH) domain. Upon activation, Btk translocates from the cytoplasm to the plasma membrane due to the production of PIP3 by phosphatidylinositol 3-kinase(PI3K) in the membrane.
The beta adrenergic receptor (βAR) is one of the most important targets for the treatment of hypertension and heart failure. Screening for βAR modulators can be achieved using Transfluor® technology. This assay principle makes use of the recruitment of β-arrestin molecules to activated receptors and their traffic within the cell and is universally applicable to GPCR activation.
G protein-coupled receptors (GPCRs) comprise one of the largest protein families encoded by the human genome and are a target for approximately 40% of all approved drugs [Eglen et al., 2007]. GPCRs sense molecules outside the cell and activate intracellular signal transduction pathways which ultimately lead to different cellular responses, most of which are linked to calcium signaling.
The cell cycle consists of four distinct phases: G1-phase, S-phase, G2-phase (collectively known as interphase) and M-phase. During the M-phase two tightly coupled processes occur: mitosis, in which the cell's chromosomes are divided and distributed to two new daughter cells, and cytokinesis, in which the cell's cytoplasm divides forming two distinct cells.
The different stages of cell cycle can be discriminated by determining the amount of DNA in each cell using a DNA stain. DNA synthesis during S-phase leads to an increase in DNA stain intercalation with an accompanying increase in fluorescence intensity. In G2/M-phase the cells will fluoresce with twice the intensity as in G1-phase. During the S-phase, the amount of DNA constantly increases, due to DNA-replication.
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.
Apoptosis or programmed cell death is the most common form of eukaryotic cell death and normally occurs during development, aging and as a homeostatic mechanism to maintain cell populations in tissues [Kerr 1972]. Cells undergoing apoptosis display profound structural changes, including a rapid blebbing of the plasma membrane and nuclear disintegration.