Fig. 2: Development of an in vitro gametocytocidal drug assay using NF54/iGP1_RE9H^ulg8 gametocytes.

Assay setup and validation. A Schematic illustrating the workflow used to produce synchronous NF54/iGP1_RE9H^ulg8 gametocytes and stage-specific gametocytocidal compound testing in a 96-well plate format. Gametocytes are exposed to compounds at the desired day of gametocyte development and gametocyte viability is determined 72 h later via RE9H-catalysed bioluminescence readout. Shld, Shield-1; GlcN, glucosamine, GlcNAc, N-acetylglucosamine. B Z‘ scores calculated from the RLUs obtained from four untreated (0.1% DMSO) and four treated (50 μM MB) samples (technical replicates) routinely included on each drug assay plate. Thick horizontal lines represent the mean Z‘ scores calculated from n = 6 (stage III and IV) or n = 8 (stage V) different assay plates, error bars indicate the s.d. and individual values are represented as black dots. C Dose-response curves for reference antimalarials (ART, CQ) and experimental compounds (KDU691, puromycin, MB) tested against synchronous NF54/iGP1_RE9H^ulg8 gametocytes at three different stages of development. Values on the y-axis represent RLUs normalized to the mean signal emitted from cells exposed to the lowest drug concentration, obtained from n = 3 (stage III and IV) or n = 4 (stage V) biological replicates (mean ± s.e.m.). IC₅₀ values are shown in Table S1.