At various time points, caspase activation was determined. colspan=”1″>?
Inactive078.973.467.262.057.850.350.045.5Low activity0C519.523.425.622.117.214.317.520.5Semiactive5C101.02.65.58.410.114.312.013.3Active10C200.30.31.36.812.714.015.614.9Very active>200.30.30.30.62.37.14.95.8 Open in a separate window ATP, adenosine triphosphate. Drug Activation of Apoptosis We were interested in determining which small molecules induce cell death through the apoptotic pathway. Apoptosis is usually often measured by detecting the activation of the caspase proteases. The challenge with Rabbit Polyclonal to GPR82 this analysis is the transient and short-lived activation of these enzymes. If a caspase activation assay is usually applied to the cells too early or after the cells are lifeless and apoptosis is usually complete, the assay result will be negative, suggesting no caspase activation and therefore no apoptosis. The windows Lactacystin of caspase activation may simply have been missed, therefore resulting Lactacystin in a false-negative result. We set out to determine whether we could use the real-time cell viability assay to determine an Lactacystin optimal windows of time, in which to multiplex a caspase activation assay to prevent missing the apoptotic windows. The real-time cell viability assay was added to cells, and luminescence was monitored every 4?h for 48?h after drug treatment. A caspase activation assay was multiplexed with the real-time cell viability assay at multiple time points throughout the time course (Fig. 5). Terfenadine resulted in significant cell death within the first 4?h of treatment. The caspase activation in these cells peaked around 4?h, which corresponds well with the real-time measurement of cell viability. Cell viability was unaffected by doxorubicin at these early Lactacystin time points, and correspondingly, there was no caspase activation within the first 4?h. In contrast, the windows of caspase activation induced by doxorubicin began around 20?h, which corresponded with a decrease in cell viability, whereas caspase activation induced by terfenadine was no longer detectable at 24?h. These two drugs show the importance of being able to target the caspase activation windows since the timing of apoptosis can differ significantly with different drugs. In both cases, when cell viability reached 50% of control cells, the caspase activation windows could be detected. As an added benefit, the luminescent caspase assay was multiplexed directly on the wells made up of real-time cell viability assay. Because the signal from the cell viability assay immediately decreases when the cells are lysed, a luminescent assay with a lytic component can be multiplexed without the need for spectral filters. The lysis component in the caspase assay killed the cells, which immediately decreased the real-time cell viability signal, and the remaining luminescence at the next read was from the caspase assay. Open in a separate windows Fig. 5. Timing of caspase activation. THP1 cells were grown in media made up of the real-time cell viability reagents and treated with 20-M terfenadine or 1-M doxorubicin. Cell viability was monitored every 4?h. At various time points, caspase activation was decided. Relative caspase activity and normalized cell viability were calculated by dividing the values from drug-treated samples by the vehicle Lactacystin control values. Doxorubicin treatment: cell viability (), caspase activation (). Terfenadine treatment: cell viability (), caspase activation (). Discussion Innovative technologies that allow drug discovery efforts to become more streamlined, affordable, and useful are needed. We describe a new cell viability assay that allows more detailed analysis of drug effects with time through a standard plate-based luminescence reading. This assay utilizes two components, a luciferase enzyme and prosubstrate, which are added to cell culture media. There is no need for cell engineering and the components can be combined with the cell suspension or drug dosing to avoid additional plating actions. The real-time cell viability assay allowed us to perform many unique analyses that are currently more laborious, expensive, and inconvenient. This assay correlated well with the number of viable cells in the well as reflected by increasing signals in proliferating cells and static signals in nondividing primary cell lines. The ability to distinguish these growth profiles indicates that this assay could be used to examine cell treatments that lead to differential cell growth and not only cytotoxicity. The assay also detected drug-induced cell death immediately. This temporal analysis of drug effects allowed fast-acting drugs (e.g., digitonin) to easily be distinguished from slow-acting drugs (e.g., thapsigargin). Being able to monitor the drug effect as many.