EC50 analysis

Adapted from Raz et al (1997) Acta Trop 68:139; see also, Baker et al (2011) Mol Biochem Parasitol 176:55

What you should end up with…EC50 plateProtocol

  1. Place 200 µl media into each well in column 1, and 100 µl into all the wells in columns 2-11
  2. Dilute the selective agent to twice the required high concentration and place 200 µl into each of the wells in column 12
  3. 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
  4. Dilute  your wild-type and test T. brucei cell lines to 4×103 cells per ml
  5. Place 100 µl cells into each well in columns 2-12 (final concentration of 2×103 cells per ml)
  6. Place at 37°C in a 5% CO2 incubator for ~66 hours

After 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

NB: after the final 6 hour incubation, plates can be placed at -20C and analysed the next day, if necessary

Analysis

Process your data in Excel (see analysis template); the output from the reader needs to be converted into ‘% inhibition’ and reorganised for GraphPad Prism.

Using the ‘Analysis’ tab in Graphpad Prism…

  • Transform concentrations to Log10
  • Normalise so that the smallest ‘% inhibition’ is set at 0% and the largest is set to 100%
  • Non-linear regressionconstraints, bottom=0 and top=100; comparison, Log10(EC50), using an F-test at 0.05; equation, ‘Sigmoidal dose response (variable slope)’.
  • This gives a final chart that you can edit and a table of results, including EC50 and Hill slope (gives rate of kill)