Determination of T. gondii growth inhibition and EC50s.

T. gondii RH strain tachyzoites expressing the firefly luciferase gene were used to identify Malaria Box molecules with antitoxoplasma activity and determine their EC₅₀s. Details regarding the generation of transgenic parasites expressing luciferase and standardization of the luminescence assay are provided in the supplemental material. The assays were set up in a 96-well plate format, and 100 µl of culture medium containing 5 × 10³ parasites was inoculated into each well containing a confluent monolayer of HFF cells, preseeded with 100 µl of culture medium containing Malaria Box compounds either at 10 µM (for growth inhibition studies) or in serial 2-fold dilutions ranging from 10 to 0.01 µM (for EC₅₀ determination). Each plate also included a standard drug as a positive control (usually atovaquone at 1 µM) and 1% DMSO as a negative control. Inhibitor treatment was done in triplicate for growth inhibition and in duplicate for EC₅₀ determination. The controls were set up as four replicates each. After 48 h of growth under optimal conditions, 150 µl of culture medium was removed and 50 µl of 2× luciferase assay reagent (Promega) was added and mixed well. The plates are immediately read with a VarioScan plate reader (Thermo Fisher, United States). The raw luminescence readings were then processed by using Microsoft Excel spreadsheets for calculation of percent growth inhibition and EC₅₀ estimation.