Reproducibility of Metabolic Inhibitor Assays

To investigate the reproducibility of each assay, cells were plated into sterile 96-well plates at a concentration of 500 cells/well and allowed to attach for 24 hours before exposure to varying concentrations of 3-bromopyruvate, lonidamine or 2-deoxyglucose. Internal triplicates for each concentration were included in each experiment. After a further 24- or 72 hours incubation period, cell enumeration assays were performed. From the data obtained after four independent experiments the concentration of each inhibitor which inhibits 50% cell viability (IC₅₀) as measured by each enumeration assay with triplicate repeats was determined using GraphPad Prism® version 4.0 for Windows (GraphPad Software, San Diego California USA, www.graphpad.com) and the variability of the IC₅₀ concentrations was compared.