ResourceLife Sciences
Design and Validation of Bioluminescent Assays for 3D Cell Culture Models
9 Sept 2014This poster demonstrates the development of an improved reagent formulation for bioluminescent detection of ATP for measuring cell viability.
This poster demonstrates the development of an improved reagent formulation for bioluminescent detection of ATP for measuring cell viability.
The CellTiter-Glo® 3D Cell Viability Assay is a homogeneous method to determine the number of viable cells in 3D cell culture based on quantitation of the ATP present, which is a marker for the presence of metabolically active cells. This ready-to-use reagent is based on the original CellTiter-Glo® Luminescent Cell Viability Assay chemistry and eliminates the need to combine buffer with lyophilized substrate when preparing reagent. The CellTiter-Glo® 3D Cell Viability Assay is formulated with more robust lytic capacity and is designed for use with microtissues produced in 3D cell culture. The homogeneous assay procedure involves addition of a single reagent (CellTiter-Glo® 3D Reagent) directly to cells cultured in serum-supplemented medium. Cell washing, removal of medium and multiple pipetting steps are not required. This assay is compatible with multiwell-plate formats, making it ideal for automated high-throughput screening (HTS). The CellTiter-Glo® 3D Assay has been used successfully with 3D microtissue cell culture produced via hanging-drop plates, ultra-low attachment plates, Matrigel®-coated plates, agarose-coated plates, cultures suspended in methylcellulose and Alvetex® plates. Features & Benefits: Robust Penetration into Microtissues - Improved lytic capacity allows use over a broad range of microtissue sizes compared to other viability assay methods. Ready-to-Use Reagent - No mixing of components required; simply thaw, equilibrate to room temperature and "add-mix-incubate-measure". Convenient for HTS applications. Fast Results - Data can be recorded in 30 minutes or less after adding reagent, quicker than when using colorimetric or fluorometric viability assays. Superior Sensitivity - The signal-to-background ratio of this assay applied to microtissues is much greater than that of standard colorimetric and fluorometric assays. Flexible Format - The assay can be used with various multiwell formats (96-well and regular or low-volume 384-well). Data can be recorded by luminometer, CCD camera or other imaging devices capable of reading luminescence in multiwell plates. Glow-Type Signal - Stable luminescent signal half-life >3 hours, depending on cell type and culture medium used, allows batch mode or consecutive processing of multiple plates. Applications: Cell viability assays with 3D microtissue cell culture or standard cell culture samples. Cytotoxicity assays with 3D microtissue cell culture or standard cell culture samples.
The CellTox™ Green Cytotoxicity Assay measures changes in membrane integrity that occur as a result of cell death. The assay is intended to assess cytotoxicity in cell culture after experimental manipulation. The assay system uses a proprietry asymmetric cyanine dye that is excluded from viable cells but preferentially stains the DNA from dead cells. When the dye binds DNA released from cells, its fluorescence properties are substantially enhanced. Viable cells produce no appreciable increases in fluorescence. Therefore, the fluorescence signal produced by the binding interaction with dead cell DNA is proportional to cytotoxicity. The CellTox™ Green Dye is non-toxic to cells, and the signal remains constant after exposure of 72 hours, making it ideal for determining toxic effects of treatments throughout an extended exposure or as an endpoint determination. Features - Benefits • Accurate Cytotoxicity Determination: The CellTox™ Green Dye stably binds DNA of cells that have lost membrane integrity throughout 72-hour exposure and won’t underestimate cytotoxicity. • Kinetic Cytotoxicity Measures: Measure cytotoxicity at convenient time points from the same sample well to detect onset of toxicity with no duplication of plates. • Simple and Flexible Protocols: Add assay reagent directly to cells prior to plating or with dosing media to perform kinetic cytotoxicity measurements, eliminating a reagent dispensing step, or add diluted dye directly to cell culture wells as an endpoint add-mix-measure assay. • Multiplexing-Compatible: Get more informative data per well and reduce cell culture expenses by multiplexing with fluorescent and luminescent cell-based assays in the same well with no sample manipulation. • Easily Automated: Easily scale from 96- to 1536-well plate formats with “no-addition” or “single-addition” protocols. Applications Determine cytotoxic effects of treatments on cells in culture after long-term exposure.
High-throughput quantitation of firefly (Photinus pyralis) luciferase expression in mammalian cells is commonly performed by batch processing of 96- and 384-well plates. Steady-Glo® Luciferase Assay System(a,b,c) is designed for this purpose by providing long-lived luminescence when added to cultured cells. The homogeneous assay provides signal half-lives of over 5 hours in commonly used cell culture media without prior sample processing. Throughput rates of several thousand samples per hour may be achieved with high reproducibility under standard laboratory conditions.
The ONE-Glo™ Luciferase Assay System provides a highly sensitive, robust, homogeneous assay for detection of firefly luciferase reporter gene expression in mammalian cells. Ideally suited for high- and ultrahigh-throughput applications, the ONE-Glo™ Assay contains a new luciferase substrate, resulting in a reagent that is more stable, more tolerant to sample components, and has less odor than standard luciferase assay reagents. These features ensure that the ONE-Glo™ Assay provides robust performance and also eliminates many of the handling inconveniences experienced using other reporter assays in a high-throughput setting. Features - Benefits Simplify Your Assay Optimization: The robust performance, reduced odor, improved storage, and larger available sizes make assay optimization even easier and more efficient. Store at Room Temperature or at 4°C: The extended stability of the ONE-Glo™ Reagent at room temperature and 4°C makes it more convenient for everyday use. Improve Assay Precision: Because the ONE-Glo™ Reagent is less sensitive to mixing and dispensing conditions, reproducibility is enhanced. The assay is ideal for use in high-density (384- and 1536-well) microplates. Get a Brighter, Longer-Lasting Signal: Optimized for batch and continuous-process handling, the extended bright light output of the ONE-Glo™ Assay allows high sensitivity, especially when extended incubation is required prior to reading results. Reduce Unwanted Effects from Sample Components: The novel chemistry used in the ONE-Glo™ Assay is less sensitive to culture media, phenol red, and luciferase inhibitors than other luciferase assays.
