Evaluation of P. falciparum growth inhibition using the standard [3H]hypoxanthine incorporation assay by filtration.

Asynchronous cultures (ca. 80% rings) of infected red blood cells (iRBCs) at 0.5% parasitemia and 2% hematocrit were incubated for 24 h in 96- or 384-well plates (100 μl and 25 μl, respectively) with drugs that had previously been dispensed from a dimethyl sulfoxide (DMSO) stock using an Echo liquid handler (Labcyte, Sunnyvale, CA, USA). The incubation culture medium contained 5 μM hypoxanthine. Then, 0.2 μCi (8 μl for the 96-well format) or 0.1 μCi (4 μl for the 384-well format) of a stock solution of [³H]hypoxanthine (0.025 μCi/μl) in RMPI 1640 was added to all the wells and the plates were incubated for an additional 24 h. Then, the plates were frozen. After that period, the plates were thawed and harvested on Filtermat A glass fiber filters (PerkinElmer) using a Cell Harvester96 cell harvester (Tomtec, CT, USA). For the 384-well plates, parasitized cultures were manually transferred to 96-well plates to undergo harvesting. The filters were dried and melted on MeltiLex scintillator sheets (PerkinElmer) to determine the incorporation of [³H]hypoxanthine. Radioactivity was measured using a MicroBeta plate counter (PerkinElmer).

Data were normalized to the level of incorporation of the positive control, consisting of iRBCs without drug (0% inhibition), and the negative control, consisting of iRBCs exposed to 25 μM artesunate (100% inhibition). All solutions were dispensed into plates using Multidrop Combi dispensers (Thermo Scientific, Waltham, MA, USA). Fifty percent inhibitory concentration (IC₅₀) values were determined using Excel software (Microsoft, Redmond, WA, USA) and fitted with Grafit (version 7) software (Erithacus Software, Horley, United Kingdom) with a 2-parameter equation.