Luciferase Assay to Study Efficacy of Antiparasitic Chemicals Against Parasites

To assess the efficacy of the inhibition of a compound of interest on Toxoplasma growth, culture human foreskin fibroblasts in 96-well microplates, for at least 7 days before inoculating each well of cells with 150 microliters of parasites, as demonstrated, for four hours at 37 degrees Celsius and 5% carbon dioxide.

During the incubation, prepare the compound of interest at eight different concentrations in a 12-walled reservoir by serial dilution.

Dilute the compounds in a 12-well reservoir at a 1:3 dilution by pipetting each mixture up and down several times with a 1-milliliter pipette.

At 4 hours post-infection, replace the medium in each well from columns 2 through 9 with 150 microliters of medium supplemented with each dilution of compound, leaving the first column filled with regular medium to serve as the non-treated control. Then, return the cell cultures to the cell culture incubator for 96 hours, and measure the luciferase activity for each well to determine the percentage of growth inhibition in the cultures at each concentration of compound.

To calculate the normalized luciferase activity as a percentage, divide the average luciferase activity for each compound concentration by the average luciferase activity derived from the non-treated parasites.

Then, use an appropriate graphing software program to plot the normalized luciferase activities against the individual compound, and to calculate the half-maximal inhibitory concentration for each compound.