Equilibration Period after Dispensing Cells

An equilibration period is often used after dispensing cells to allow uniform attachment before addition of test compound. The length of equilibration can be easily determined and optimized to reach an acceptable window between positive and negative control responses. In some cases, the equilibration step is eliminated when compounds are dispensed immediately after seeding cells and in other cases the test compounds may have already been dispensed into the assay plates prior to dispensing cells.

6.2.8 Concentration of Compound Tested

The available concentration of a compound may determine whether it is toxic to cells. The concentration of a test compound available to cells may not be the same as the final concentration added to the sample if the toxin is bound by albumin present in serum used to supplement the medium. Chemical compounds also may bind to plastic surfaces, become concentrated inside cells, be chemically converted by modifying enzymes in the cytoplasm, or actively pumped out of cells.

Because chemical libraries contain compounds with diverse physiochemical properties, it may be a challenge to choose an initial concentration to test. Many screening campaigns testing small molecule libraries typically use 10 pM compound delivered in 0.1% to 0.2% DMSO. Although this represents a good starting point, using quantitative HTS (qHTS; Inglese et al. 2006; Xia et al. 2008) presents a tremendous advantage for testing a range of concentrations during primary screening. The qHTS approach that typically uses the 1536-well format provides much additional data and can overcome many of the limitations of screening at a single toxin concentration.

6.2.9 Length of Exposure to Test Compound

Determining the appropriate length of exposure of cells to test compounds can be a balance between the limits of nutrient depletion and the time needed by cells to respond to an experimental treatment. The toxicity of a compound to a population of cells and the magnitude of response can be related to both the concentration and length of exposure (Riss and Moravec 2004). Figures 6.1 and 6.2 show that the duration of exposure of cells to test compounds can directly affect whether the population of cells will be scored as viable, dead or apoptotic. For instance, some test compounds can be exposed for a few hours with no apparent effect on viability, but induce markers of apoptosis.

One of the main challenges in designing cytotoxicity and apoptosis assays for screening is determining an appropriate length of compound exposure. Difficulties arise because different classes of

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Tamoxifen

FIGURE 6.1 Characterization of toxic effects of tamoxifen on HepG2 cells measured using a luminescent ATP assay as an indicator of cell viability. Times in the legend indicate durations of tamoxifen exposure. Loss of viability is dependent on concentration of toxin and duration of exposure. (Source: Modified from Riss, T.L. et al. 2005. Selecting cell-based assays for drug discovery screening. Promega Cell Notes 13, 16-21.)

Tamoxifen

FIGURE 6.1 Characterization of toxic effects of tamoxifen on HepG2 cells measured using a luminescent ATP assay as an indicator of cell viability. Times in the legend indicate durations of tamoxifen exposure. Loss of viability is dependent on concentration of toxin and duration of exposure. (Source: Modified from Riss, T.L. et al. 2005. Selecting cell-based assays for drug discovery screening. Promega Cell Notes 13, 16-21.)

—•— 0 hour --o-- 0.5 hour —□- 2 hour —A— 6 hour -o- 24 hour

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Tamoxifen

FIGURE 6.2 Characterization of toxic effects of tamoxifen on HepG2 cells measured with the bis-Z-DEVD-R110 substrate in a fluorescent caspase-3/7 assay as an indicator of apoptosis. Times in the legend indicate duration of tamoxifen exposure. Activation of apoptosis is dependent on concentration of toxin and duration of exposure. Activation of caspase activity in time zero sample occurred during incubation with fluorogenic reagent. (Source: Modified from Riss, T.L. et al. 2005. Selecting cell-based assays for drug discovery screening. Promega Cell Notes 13, 16-21.)

—•— 0 hour --o-- 0.5 hour —□- 2 hour —A— 6 hour -o- 24 hour

0 40 80 120

Tamoxifen

FIGURE 6.2 Characterization of toxic effects of tamoxifen on HepG2 cells measured with the bis-Z-DEVD-R110 substrate in a fluorescent caspase-3/7 assay as an indicator of apoptosis. Times in the legend indicate duration of tamoxifen exposure. Activation of apoptosis is dependent on concentration of toxin and duration of exposure. Activation of caspase activity in time zero sample occurred during incubation with fluorogenic reagent. (Source: Modified from Riss, T.L. et al. 2005. Selecting cell-based assays for drug discovery screening. Promega Cell Notes 13, 16-21.)

compounds in a diverse library will exhibit a broad range of effects on the model system chosen and may require longer exposure periods to elicit responses. For instance, long term exposure to sublethal doses of compounds may eventually result in toxicity (O'Brien and Haskins 2006) that would not be detected using a shorter incubation period.

Time course experiments can be performed using different classes of control toxins to determine the length of exposure necessary to induce apoptosis or result in necrotic cell death. A toxin with properties that disrupt cell membranes will result in rapid necrotic cell death. Other chemicals may not become toxic until after conversion by modifying enzymes in the cytoplasm. In many cases, subpopulations of cells at different stages of the cell cycle may undergo cell death at different times.

All cells undergoing apoptosis in vitro eventually undergo secondary necrosis. This results in disruption of the cell membranes and spilling of cytoplasmic components into the surrounding medium. This is in contrast to the in vivo situation in which early engulfment of apoptotic cells by macrophages occurs. Caspase activity (as a marker of apoptosis) may increase initially in a population of cells induced to undergo apoptosis, but eventually decline after secondary necrosis occurs as the enzymes become degraded. One of the benefits of understanding the sequence of apoptotic events in vitro is knowing that assays to measure markers of cell viability or necrosis can be used to detect the end result of apoptosis. Regardless of whether the mechanism of cell death is apoptosis or necrosis, after long term exposure all the cells will be scored as dead with a cell viability assay.

Two general approaches may overcome uncertainty in establishing the duration of incubation. One approach that can be useful when testing a single concentration of test compound is to use multiplexing to measure more than one assay endpoint. Multiplexing can help confirm or rule out a positive response (hit) by measuring two independent markers of the same event or two different events (cell viability and apoptosis). Another and perhaps more powerful approach is to use qHTS to test a broad range of compound concentrations. The rationale is that a range of concentrations is more likely to demonstrate toxicity during a defined incubation period, whereas testing only a single standard dose may not show an effect.

For example, if a cell viability marker assay indicated a population of cells was dead after 48-hr incubation with 10 pM of a test compound, it would be impossible to determine whether cell death resulted from apoptosis. It is possible the cell population underwent apoptosis soon after chemical treatment followed by secondary necrosis, and the apoptosis marker (caspase activity) subsequently degraded in a pool of cell debris and culture medium. A relatively high concentration of test compound may induce apoptosis in a population of cells in less than an hour, but a much lower concentration of the same compound may not induce apoptosis for several hours. In cases where lower concentrations of a chemical induce apoptosis only after longer incubation periods, testing a broad range of concentrations of the same compound may indicate that 0.01 pM induces apoptosis not observed with higher concentrations. Dosage and length of exposure clearly impact the result of endpoint assays.

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