Synergy H4 Optimized Instrument Settings.
After removal from the growth flask, the cells were resuspended in the previously described serum-free, glucose-free medium. The fluorescent signal was then read using the aforementioned Synergy H4 settings. The microplate was then equilibrated to room temperature for approximately 15 minutes.
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The resulting luminescent signal was then read on the Synergy H4. Luminescent or fluorescent values from wells containing media, treatments, and assay reagents were subtracted from raw values detected from cell containing wells.
Where Value T equals the background subtracted value from wells containing compound, and Avg Value U equals the average value from background subtracted basal wells containing no compound. Per Figure 3, ATP Detection Assay data, ATP production, as a function of the detected luminescent signal, substantially decreases in the hepatocyte cells in the presence of antimycin.
The lack of significant change in cytotoxicity data proves that toxicity from antimycin is due to the compound impeding mitochondrial function, and not a result of primary necrosis.
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ATP production and cytotoxicity data are similar in hepatocytes grown with and without glucose. Primary hepatocytes assayed in either high glucose or no glucose in the presence of increasing concentrations of the mitotoxicant antimycin show significant decrease in ATP production as expected. ATP production in the HepG2 cells Figure 4 reveals a sharp contrast between cells grown in glucose media and those in non-glucose media. Mainly, those HepG2 cells grown in non-glucose media were forced to revert to produce ATP via mitochondrial oxidative phosphorylation, and therefore behaved similarly to the hepatocyte cells, while HepG2 cells grown in glucose media produced ATP via glycolysis, therefore the known antimycin toxic properties on mitochondria were masked in a classic Crabtree Effect.
Cancerous hepatocytes HepG2 grown in no glucose media display a similar ATP decrease, while those grown in high glucose media have very little ATP decrease, thus representative of the Crabtree Effect.
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Because of their ease of use, and availability, highly proliferative tumor-derived immortalized cell lines have become the model of choice for mitochondrial toxicity studies. This has led to possible false conclusions regarding lead compound toxicity due to the cells propensity to derive energy from glycolysis rather than through oxidative phosphorylation in the mitochondria.
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