ATP articles and MTS reductase activity, exposed a prevalent and vital artifact in that, below situations of cell cycle arrest, the assumed linear romantic relationship amongst assay signal and cell amount breaks down. The typical volume of ATP or MTS lowering activity per cell is significantly enhanced. This enhance correlates with, and will be explained by, drug induced increases in per cell cell dimension and consequently mitochondrial material. In comparison, estimation of viable cell amount by using complete DNA fluorescence was much less prone to deviation in the real cell number. Prior scientific studies have also reported differences in cell variety determination in between DNA quantification and metabolic process based mostly assays . On the other hand, treatment options that substantially transformed the common DNA content material per cell by inducing mitotic arrest and or endoreduplication also led to an underestimation during the percent change in cell number using the CyQuant assay.
On the whole, alterations in cellular ATP content and MTS activity resulted in 1 of two forms of deviations among cell variety and ATP MTS assay. Very first, there have been cases exactly where Emax was considerably decreased, i.e. particularly shallow dose response curves. Such as selleck you can check here gemcitabine lowers ATP signal for HT29 cells by somewhere around twenty , when in actual fact cell amount is reduced by 80 relative to manage. The other condition is exemplified by etoposide, wherever the EC50 is best shifted however the dose response curves converge to comparable Emax at a sufficiently substantial concentration. This could lead to underestimation of antiproliferative potency by ten fold or even more.
VX 680 presents an intermediate case, wherever there exists a biphasic ATP curve with an intermediate plateau corresponding to an elevated ATP cell ratio, followed by a second decline. Curves like this fit poorly, if in any way, to your typical 4 parameter selleck more info here model and will result in incredibly inconsistent success with regards to the two EC50 and Emax. These unique response profiles is usually explained by the mechanisms of action of those compounds: There exists a biphasic cell cycle dose response to a lot of these drugs. First, on target antiproliferative or cytostatic responses coincide with all the reduction in absolute cell variety but small cell death. Beneath these situations the arrested cells maximize in dimension and mitochondrial content, and correspondingly, the amount of ATP per cell increases. At greater drug concentrations the population phenotype may well turned out to be significantly less exclusively cytostatic, based on cell line and treatment method time.
With escalating late apoptotic fraction the common MitoTracker intensity and ATP and MTS per cell declines.