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Cell Spreading BioApplication and HCS Reagent Kit |
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Description
Cell attachment and spreading are multifactorial events mediated by interactions between adhesion molecules on the cell surface and underlying extracellular matrices. Regulation of these events is central to a number of physiological processes including cancer cell metastasis, inflammation, and angiogenesis. Rather than measuring cell-ligand binding constants, the efficacy of drugs that modulate these functions can be measured in single cells by monitoring attachment and spreading on a tissue-specific substrate. Such morphological-based analyses can be timeconsuming if done manually, and general image-analysis packages can be difficult to program to get the most information on a biological event. The Cell Spreading BioApplication and HCS Reagent Kit provide a rapid, automated, functional assay of the regulatory and structural proteins needed for morphological changes in the cell (Figure 1). The Cell Spreading HCS Reagent Kit provides high-content screening (HCS) quality fluorescence reagents and a validated, optimized protocol necessary to quantify the spreading of cells by directly measuring their areas using a fluorescent cytoskeletal marker, rhodamine-conjugated phalloidin (Figure 2). The assay is performed on cells grown on high-density microplates or microscope slides. In addition to fluorescent phalloidin, the kit also includes the nuclear dye Hoechst to quantitate nuclei and distinguish cell colonies or aggregates. A control compound that prevents cells from spreading without affecting adherence to the underlying substrate is also included in the kit.
Cells can be imaged and analyzed on a Thermo Scientific Cellomics ArrayScan HCS Reader with the Cell Spreading BioApplication software, resulting in quantitative outputs of cell morphology (Figure 3). The Cell Spreading BioApplication utilizes two fluorescence channels to identify the cell nucleus and the cytoplasm. Objects identified using the cytoplasmic stain are recognized as either monodispersed cells if they contain a single nucleus or as a colony if they contain multiple nuclei. Contiguous colonies are considered a single cytoplasmic object and so the number of cytoplasmic objects is often less than the number of cells (nuclei) in the field. The BioApplication measures the area and perimeter of single cell or colonies of cells during spreading. The user can set an upper limit on the number of cells per colony. Figure 3 below shows data plotted from a cancer cell spreading experiment. Cells stained with the Cell Spreading HCS Reagent Kit are efficiently measured by the BioApplication, providing a quantitative assessment of the state of the cell’s cytoskeleton. The combination of the Cell Spreading BioApplication and HCS Reagent Kit provides an automated method for screening compounds for their ability to modulate cell spreading processes.
Features
- The BioApplication and HCS Reagent Kit are versatile and widely applicable to a variety of cell types
- Cell-based assay yields an excellent window for screening compounds based on cell function, rather than ligand binding
- Validated protocol for sample preparation/processing requires approximately 1.5 hours postcompound incubation
- Assay validated in standard microplates for streamlined processing
- Utilizes fluorescent reagents and requires no cell lysis, purification, or filtration steps; and no handling of radioactivity
- Prepared cells may be analyzed directly via standard fluorescence microscopy; automated quantification of cell spreading is achieved using the ArrayScan HCS Reader
- The Reagent Kit and BioApplication together provide a complete solution for studying cell spreading
Click the link below to view available product brochure
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