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Chapter: Basic Concept of Biotechnology - Animal Biotechnology

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Cell Viability Assays that Measure ATP and Metabolic Capacity

CellTiter-Blue Cell Viability Assay (resazurin), CellTiter-Glo Luminescent Cell Viability Assay.

Cell Viability Assays that Measure ATP

 

CellTiter-Glo® Luminescent Cell Viability Assay

 

The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture. Detection is based on using the luciferase reaction to measure the amount of ATP from viable cells. The amount of ATP in cells correlates with cell viability. Within minutes after a loss of membrane integrity, cells lose the ability to synthesize ATP, and endogenous ATPases destroy any remaining ATP; thus the levels of ATP fall precipitously. The CellTiter-Glo® Reagent does three things upon addition to cells. It lyses cell membranes to release ATP; it inhibits endogenous ATPases, and it provides luciferin, luciferase and other reagentsnecessary to measure ATP using a bioluminescent reaction. The unique properties of a proprietary stable luciferase mutant enabled a robust, single-addition reagent. The "glow-type" signal can be recorded with a luminometer, CCD camera or modified fluorometer and generally has a half-life of five hours, providing a consistent signal across large batches of plates. The CellTiter-Glo® Assay is extremely sensitive and can detect as few as 10 cells. The luminescent signal can be detected as soon as 10 minutes after adding reagent or several hours’ later, providing flexibility for batch processing of plates.

 

Cell Viability Assays that Measure Metabolic Capacity

 

CellTiter-Blue® Cell Viability Assay (resazurin)

The CellTiter-Blue® Cell Viability Assay uses an optimized reagent containing resazurin. The homogeneous procedure involves adding the reagent directly to cells in culture at a recommended ratio of 20µl of reagent to 100µl of culture medium. The assay plates are incubated at 37°C for 1–4 hours to allow viable cells to convert resazurin to the fluorescent resorufin product. The conversion of resazurin to fluorescent resorufin is proportional to the number of metabolically active, viable cells present in a population. The signal is recorded using a standard multiwellfluorometer. Because different cell types have different abilities to reduce resazurin, optimizing the length of incubation with the CellTiter-Blue® Reagent can improve assay sensitivity for a given model system. The detection sensitivity is intermediate between the ATP assay and the MTS reduction assay.


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