Late-stage assay conditions.

For all the luminescence readouts, we used the homogeneous luciferase reporter gene assay system Steadylite Plus (PerkinElmer). A previously validated protocol was employed to lyse the cultures, and luciferase substrate was added (15). First, 25 μl medium was aspirated from the assay plates and replaced with 15 μl Steadylite, without disturbing the settled RBCs, using a Biomek FX high-throughput liquid-handling system (Beckman Coulter). Luminescence was measured after the relevant incubation time at room temperature using a MicroBeta Trilux multidetector luminometer equipped with a plate stacker for high-throughput applications.

The effect of the percent gametocytemia and the percent hct on the luciferase signal was assessed in 384-well white luminescence plates (Culturplate; PerkinElmer) after 72 h of incubation under standard conditions. Sixteen replicate wells were seeded with day 8 gametocytes at a range of hct percentages (0.1, 0.25, and 0.5%) and gametocytemia percentages (1.5, 3, 6, and 12%). After the incubation, the luciferase signal was read in the plates at regular intervals from 1 h to 6 h 45 min after the addition of Steadylite.

The consistency of the gametocytocidal compound potency data generated by the assay was tested by measuring the 50% inhibitory concentration (IC₅₀) of puromycin at multiple time points after the addition of the luciferase kit. The Z′, a measure of assay quality and reproducibility for predicting suitability for screening, was determined based on 0.4% dimethyl sulfoxide (DMSO) and 5 μM puromycin as negative and positive controls, respectively (24).