Gametocyte Inhibition Assay for P. falciparum

Gametocytes were induced from asexual P. falciparum NF54 parasites as described before^{61 (link)}. The parasite lactate dehydrogenase (pLDH) assay was performed as previously described^{59 (link),62 (link)}. Early and late stage P. falciparum gametocyte cultures (2% haematocrit, 5% gametocytaemia) were treated with compounds at 1 and 5 μM (primary dual-point primary screening), with methylene blue (5 µM) as positive control for inhibition. Gametocytes were treated under drug pressure for 72 h at 37 °C, followed by drug washout with culture medium and remaining pLDH activity was determined 72 h later by addition of Malstat reagent and absorbance measured with a Multiskan Ascent 354 multiplate scanner (Thermo Labsystems, Finland) at 620 nm. The percentage of viability was plotted as a function of drug concentration and curve fitting was obtained by non-linear regression analysis using a four-parameter logistic method. A full dose-response evaluation was performed for hit compounds against early and late stage P. falciparum gametocytes for three independent biological replicates. Assay performances were evaluated with average %CV at 4.54 and Z-factors at >0.5.