Applicazioni più innovative
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Spheroid Imaging | Phenotypic Assays | Live Cell Kinetic Assays | Biotherapeutics | Biofuel Research
A major trend in cell-based assays is to increase the physiological relevance of the data generated. Tissue culture in three dimensions (3D) is at the forefront of these efforts to mimic organotypic responses. The most popular formats of 3D cell culture involve scaffolds built in microplate wells using a variety of materials such as hydrogels, collagen, electrospun fibers and others; and spheroids consisting of cell aggregates (~ 103 - 104 cells) that can be formed using hanging drop microplates or ultra low absorbance coated microplates. The Cytation Cell Imaging Multi-Mode Reader facilitates all popular 3D cell culture models: particularly its automated digital microscopy capability enables live cell kinetic assays involving spheroids. Furthermore, the MultiFlo FX Multi-Mode Dispenser provides a means to automate the generation of 3D cultures and the performing of assays.
These publications demonstrate the utility of both PMT- and CCD-based optics in the Cytation for 3D cell culture assays:
- AACR 2015 Presentation: Phenotypic and MoA Analysis of Anti-Metastatic Molecules using 3D Spheroid-Based Tumor Invasion Assays, Kinetic Microplate Detection, and Cellular Microscopy
- Quantification of MMP Activity and Inhibition in a 3D Tumor Invasion Model
- Cellular Analysis of 3D Spheroid-Based Tumor Invasion Assays
- Inhibition of Hypoxic Tumor Cells using a Three-Dimensional Spheroid Model
- Live Cell Hypoxia Determination in a 3D Spheroid Model
- Automated Cell Dispensing and Image-Based Spheroid Formation Tracking
A phenotype is the physical expression of a genotype. In phenotypic assays, a phenotype of cells is typically caused or suppressed by stimuli. Common phenotypes that are assayed are the production of reactive oxygen species and hypoxia using fluorescent probes, but may also include cell morphology changes, migration, apoptosis or necrosis. These assays have once again gained prominence in drug discovery due to no requirement of mechanism of action knowledge. Many diseases involve complex biology which is not centered on a single protein that can be targeted for disease intervention, thus drug target-based assays are being replaced in many cases with phenotypic assays in early drug discovery processes. Automated digital fluorescence microscopy using microplates is a preferred method for phenotypic assays as multi-parametric data can be obtained using a wide array of fluorescent probes to quantify phenotype changes.
The Cytation Cell Imaging Multi-Mode Reader and the MultiFlo FX Multi-Mode Dispenser are well equipped to automate workflows and perform common operations such as cell seeding into microplates, media exchanges, reagent additions and automated digital microscopy common to phenotypic assays as described in these publications:
- Measuring Real-time Cellular Respiration and Mitochondrial Function using Fluorophoric Biosensors
- Label-Free Phenotype MicroArrays™ Analysis of Cellular Energetics and Apoptotic Activity using Microplate Reading and Phase Contrast Imaging
- Phenotypic and Epigenetic Mechanism of Action Determinations of Histone Methylase and Demethylase Inhibitors using Digital Widefield Microscopy
- Use of Phase Contrast Imaging to Track Morphological Cellular Changes due to Apoptotic Activity
- Investigation of Cell Migration using a High Density Cell Exclusion Assay and Automated Microplate Imager
- Induction and Inhibition Studies of Hypoxia and Oxidative Stress in Immortalized Keratinocytes
Kinetic assays performed on live cells provide real-time data of cellular responses. One of the most common live cell kinetic assays has been available for decades and involves the use of fluorogenic calcium indicators for measuring Ca2+ flux in various cells. This response is rapid requiring only one or two decades of seconds to reach maximal response and the signal decays rapidly in one or two minutes. Today there are a wide variety of live cell assays based on the use of multiple detection modalities involving both PMT- and CCD-based detection. The Synergy H1 Multi-Mode Microplate Reader and the Cytation™ Cell Imaging Multi-Mode Reader have both been designed to optimize live cell assays. Each reader can be equipped with an optional Reagent Injection Assembly for controlled compound reagent addition for fast response kinetic assays and a Gas Control unit that allows for control of O2 and CO2 levels within the detection chamber enabling long term kinetic studies under suitable conditions for cell health.
The following publications demonstrate these capabilities in BioTek instrumentation:
- Live Cell Imaging of RNA Expression
- Live Cell Imaging of GPCR Dependent Second-Messenger Systems
- A Bioluminescent Live-Cell cAMP Assay for the Pharmacological Evaluation of GPCRs
- Live Cell Biosensor Assay Used To Interrogate GPCRs
- Functional Characterization of Multi-Drug Resistance Transporters in Living Cells
The financial success of biologics, the new “blockbuster drugs,” such as recombinant erythropoietin and a number of immunotherapeutics (Rituximab (Rituxan), Trastuzumab (Herceptin), etc.) where annual revenues exceed US$5B, has spurred drug discovery organizations to pursue not only novel biotherapeutics, but also biosimilar products to successful drugs coming off patent. Antibody-Dependent Cell-mediated Cytotoxicity (ADCC), Blocking Antibody and Immunogenicity assays are common microplate assays in the biotherapeutics drug discovery and development process. BioTek's Synergy™ product line of Multi-Mode Microplate Readers, encompassing Synergy H1/H4 and Synergy Neo, are well suited to read the many non-radiometric detection variants of these assays incorporating absorbance, bioluminescence, TRF, HTRF and AlphaLISA detection modes. Furthermore, the automated pipetting station Precision Microplate Pipetting System provides semi-automation of the assay workflows suitable for the sample throughput of these assays.
These publications describe these assays and the automated workflows:
- Automation of a Homogeneous Proximity Assay for Immunogenicity Testing
- An Automated DELFIA ADCC Assay Method using a CD16.NK-92 Cell Line
- Automated Bioluminescent ADCC Reporter Bioassay Using Bioengineered Jurkat Cells
- Automated High Throughput Functional Cell-based Bioassays for EGFR Blocking Antibodies
- Automated Cellular Screening and Characterization of Therapeutic Antibodies for ADCC
The increasing cost of fuel, as well as the impact of climate change, has dramatically increased the interest in alternative fuel sources. First generation biofuels based on the use of feedstock starches converted to ethanol is currently in wide use, but is not sustainable. Second generation biofuel production using lignocellulosic materials such as switchgrass and third generation biofuels from algae offer hope for the provision of sustainable, green energy sources. Colorimetric, turbidimetric and fluorometric microplate assays are commonly used to assess methods for the economic enzymatic breakdown of the polysaccharides associated with lignocellulosic material and the selection of algal strains and conditions optimal for biofuel production.
These publications demonstrate that Synergy™ H4 is a particularly useful microplate reader for all these assays associated with biofuel research:
- Monitoring Enzymatic Glucose Production from Cellulosic Feedstock
- Enzymatic Digestion of Polysaccharides (Part I)
- Enzymatic Digestion of Polysaccharides (Part II)
- Monitoring of Algal Growth Using Their Intrinsic Properties
- Determination of Algal Cell Lipids Using Nile Red