Determination and comparison of EC50s Sample Clauses

Determination and comparison of EC50s. In order to investigate the overall effect of a compound on a particular cell line, EC50s for each experiment were determined from dose-response curves as follows. Data were normalised as percentage of untreated (control) cells before concentration (in M) was log-transformed. These were then plotted using an XY scatter graph with log concentration as the independent variable and percentage of untreated (control) cells as the dependent variable. A non-linear regression analysis was then carried out using a log inhibitor vs response – variable slope model to the following equation: 𝑌 = 𝐵𝑜𝑡𝑡𝑜𝑚 + 𝑇𝑜𝑝 − 𝐵𝑜𝑡𝑡𝑜𝑚 1 + 10(𝐿𝑜𝑔𝐸𝐶50−𝑋) ∗ 𝐻𝑖𝑙𝑙𝑆𝑙𝑜𝑝𝑒 A constraint at Y = 0 was used due to the subtraction of background readings from all toxicity data. Two different EC50s can be calculated from this data. The first one, the relative EC50, is the point halfway between the top and the bottom of the plotted curve. The relative EC50 takes into account instances where the lowest concentrations of a compound achieve lower viability than untreated cells or instances where the lowest concentrations actually enhance the viability of the cells beyond 100%. While it can be of interest to take into account these effects at lower concentrations when determining general toxicity, the comparison of 2 separate relative EC50s is difficult, due to the fact that they could be derived from largely different areas of the y-axis. However, this is not the case for the absolute EC50. The absolute EC50 is determined by calculating the point on the x-axis (the log concentration), at which the curve intersects 50% viability (relative to untreated cells) on the Y axis. Due to the desire to compare toxicity between cell lines using a fixed point of reference during our studies, only the absolute EC50s were determined. As a consequence, any further mention of EC50 will be in reference only to the absolute EC50. Furthermore, in order to differentiate between EC50s calculated following the MTT or ATP assays, the nomenclature EC50MTT and EC50ATP will be used in future chapters. During statistical analysis, EC50s were generated and maintained in their log format due to the requirement for concentration to be log-transformed when constructing a dose-response curve. It was not considered to be statistically sound to perform statistics with the antilog of the logEC50s, due to the uneven symmetry that exists when a log standard error is anti-logged. For example, an EC50 of 1 µM +/-...