Pharmaceutical companies approach drug discovery in a variety of ways. An early part of the experimental process often involves screening a large number of compounds using defined biochemical assays in an ultrahigh-throughput format.
However, the effect of a drug on an organism is complex and involves interactions at multiple levels that cannot be predicted using biochemical assays. Trying to understand this complexity has contributed to an increased use of cell-based screening assays as more biologically relevant surrogates to predict the response of the organism. In addition, at some point in the drug discovery process, predicting cellular toxicity is important.
Eukaryotic cell culture is accepted as the model system of choice to get a first approximation of toxicity. Furthermore, advances in assay chemistries and signal detection technology have allowed miniaturization of cell-based assays, making it more convenient to perform dose-response experiments during primary screens.
(1) ATP Assay of Cell Viability
The amount of ATP in cells correlates with cell viability. Within minutes after loss of membrane integrity, cells lose the ability to synthesize ATP; endogenous ATPases destroy any remaining ATP, and ATP levels fall precipitously. 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 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 and luciferase necessary to measure ATP using a bioluminescent reaction. The “glow-type” signal of the proprietary Ultra-Glo™ Luciferase can be recorded with a luminometer, CCD camera or modified fluorometer and generally has a halflife of five hours, providing a consistent signal across large batches of plates.
The CellTiter-Glo® Assay can detect as few as 15 cells Although equilibration of assay plates to room temperature is recommended before performing the assay, the assay can be completed rapidly. The luminescent signal can be detected as soon as 10 minutes after adding reagent or several hours later for batch processing of plates. Among the homogeneous viability assays, the ATP assay is the fastest to perform and can detect the smallest number of cells, making it useful for 384- and 1,536-well formats.
(2) Tetrazolium Reduction Cell Viability Assay
The CellTiter 96® AQueous One Solution Cell Proliferation Assay is the industry standard for homogeneous colorimetric cell viability assays. Viable cells convert the MTS tetrazolium reagent into a colored formazan product during a 1- to 4-hour incubation. The amount colored formazan product is directly proportional to the number of viable cells.
(3) Resazurin Reduction Cell Viability Assay
The CellTiter-Blue® Cell Viability Assay uses an optimized reagent containing resazurin, which is reduced to fluorescent resorufin in living cells. The reagent is added directly to cells in culture, incubated, and the signal is read using a multiwell fluorometer. Because different cell types have different abilities to reduce resazurin, optimizing incubation time 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. The simple, inexpensive procedure can be multiplexed with other assays to collect a variety of data. The assay provides good Z´-factor values in HTS situations as well.
(4) LDH-Release Cytotoxicity Assay
Cells that have lost membrane integrity release lactate dehydrogenase (LDH) into the surrounding medium. The CytoTox-ONE™ Homogeneous Membrane Integrity Assay is a fluorescent method that uses coupled enzymatic reactions to measure the release of LDH from damaged cells as an indicator of cytotoxicity. The assay is designed to estimate the number of nonviable cells present in a mixed population of living and dead cells. Alternatively, if a cell lysis reagent is used, the same assay chemistry can be used to determine the total number of cells in a population. The CytoTox-ONE™ Reagent does not damage living cells, and the assay can be performed directly in cell culture using a homogeneous method. The CytoTox-ONE™ Assay is fast, typically requiring only a 10-minute incubation period, and is compatible with 96- and 384-well formats. The detection sensitivity is a few hundred cells but can be limited by the LDH activity present in serum used to supplement culture medium. When automated on the Biomek® 2000 workstation, the CytoTox-ONE™ Assay gave excellent Z´-factor values.
(5) Fluorescent Caspase-3/7 Assay to Detect Apoptosis
The activity of executioner caspases such as caspase-3 and -7 is an accepted, reliable indicator of apoptosis. The Apo-ONE® Homogeneous Caspase-3/7 Assay detects caspase-3/7 activity based on the cleavage of a profluorescent DEVD peptiderhodamine 110 substrate. The Apo-ONE® Reagent is prepared by combining buffer and substrate and adding it directly to culture wells using a 1:1 ratio of reagent to medium, mixing and incubating. The reagent permeabilizes the cells to release the caspase, delivers the profluorescent substrate, and provides optimized conditions to stabilize caspase activity. Because the fluorescent R110 product continues to accumulate in the presence of active caspase-3 and -7, extending the incubation period up to 18 hours increases the signal-tobackground ratio, providing greater sensitivity. The Apo-ONE® Assay is easily scalable for HTS as long as the 1:1 ratio of reagent to medium is maintained. The detection sensitivity is in the range of several hundreds of cells but can be influenced by the length of incubation.
(6) Luminescent Caspase Assays to Detect Apoptosis
The Caspase-Glo® 3/7, 8 and 9 Assays measure caspase activity based on the cleavage of a peptide-aminoluciferin substrate. Caspase cleavage of the substrate liberates free aminoluciferin, which can be used as a substrate by luciferase to generate light. The Caspase-Glo® Reagent is prepared by combining a lyophilized substrate and buffer. The reagent is added directly to cells in culture at a 1:1 ratio of reagent to medium, mixed and incubated, and luminescence is recorded. The assay has a flexible incubation time for recording the “glow-type” luminescent signal. When steady state is reached after approximately 30 minutes to one hour of incubation, the luminescent signal of this coupled enzymatic assay is directly proportional to the amount of caspase over a broad linear range. The Caspase-Glo® Assays are the most sensitive caspase assays available. Because they are luminescent assays, fluorescent compounds will not interfere with results.
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