Fig. 2.

Schematic diagram for IC₅₀shift experiment for determining parasite tolerance to anti-malarial drugs. Parasite cultures between 0 and 2 h of age were isolated for each parasite culture tested (Fig. 1). Each respective culture was dispensed into 15 × 384-well Perkin Elmer cell carrier imaging plates. All plates were placed within their relevant incubation conditions and at each compound addition time point (identified by a bold red arrow) a single plate for each of the parasite culture conditions was removed from incubation and 5 μl of diluted compound added. The order in which the 5 μl of diluted compounds were added to the parasite culture containing plates was maintained at each time point. The parasite assay plates containing the compounds were placed back into their respective incubation conditions. This was performed for each of the 15 designated time points throughout the parasite asexual intra-erythrocytic life cycle, as indicated by each red arrow up to and including 35 h post RBC invasion. All the plates were then stained with DAPI at the 65 h post RBC invasion time point and imaged on the Opera HTS confocal imaging system. Parasite numbers were analysed as described previously (Duffy and Avery, 2012). The number of parasites were normalized to in-plate control images (5 μM puromycin or 0.4% DMSO) and expressed as a % inhibition of parasite proliferation. The compound concentration was then plotted against % inhibition of parasite proliferation. The data was analysed using nonlinear regression, sigmoidal dose response (variable slope) with no top or bottom constraints analysis. The IC₅₀ for each compound at each compound addition time point throughout the intra-erythrocytic cycle was calculated. The fold change in IC₅₀ values for each compound was calculated by dividing the IC₅₀ obtained at each subsequent time point by that obtained at the first time point of 3 h post RBC invasion. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)