This model assumes that the dose response curves has a standard slope, equal to a Hill slope (or slope factor) of -1.0. The goal is to determine the IC50 of the inhibitor - the concentration that provokes a response half way between the maximal (Top) response and the maximally inhibited (Bottom) response. response curves follow the familiar symmetrical sigmoidal shape. Non-linear regression – constraints, bottom=0 and top=100 comparison, Log 10(EC 50), using an F-test at 0.Equation: log(inhibitor) vs.Normalise so that the smallest ‘% inhibition’ is set at 0% and the largest is set to 100%.Using the ‘Analysis’ tab in Graphpad Prism… Process your data in Excel (see analysis template) the output from the reader needs to be converted into ‘% inhibition’ and reorganised for GraphPad Prism. NB: after the final 6 hour incubation, plates can be placed at -20C and analysed the next day, if necessary Place at 37☌ in a 5% CO 2 incubator for ~66 hoursĪfter 66 hours add 20 µl 0.125 mg/ml resazurin (Sigma) to each well and incubate for a further 6 hours the resultant colour change in the presence of living cells (from blue to pink) can be quantified on a plate reader: excitation, 530 nm emission, 585 nm filter cut-off, 570 nm.

Place 100 µl cells into each well in columns 2-12 (final concentration of 2×10 3 cells per ml).Take 100 µl from each well in column 12, transfer to column 11 and pipette up and down three times to mix repeat, generating a two-fold descending dilution series from column 12 to 3, inclusive.Dilute the selective agent to twice the required high concentration and place 200 µl into each of the wells in column 12.Place 200 µl media into each well in column 1, and 100 µl into all the wells in columns 2-11.Adapted from Raz et al (1997) Acta Trop 68:139 see also, Baker et al (2011) Mol Biochem Parasitol 176:55

Prism ic50