PFNF54-Pfs16-GFP-LUC gametocytes treated at early stage or late stage development with compound MMV666810 or MMV674850

Plasmodium falciparum gametocytes can often show differential drug responses depending on their stage of development. Here, the transcriptomic response of early stage (Stage II-III) and late stage (Stage IV-V) gametocytes was evaluated following treatment with chemical compounds that show differential killing activity between early and late-stage gametocytes. P. falciparum NF54-Pfs16-GFP-LUC parasites were induced to produce gametocytes by culturing in glucose-deficient media , before removing asexual parasites from the population by daily 5% D-sorbitol treatments from day 1-4 and thereafter culturing the parasites in glucose positive media and grown under stationary conditions for the entire experiment. The compounds were added to the cultures at 10x their previously determined IC50 values on day 5 (early-stage gametocytes) and 10 (late-stage gametocytes) and samples taken at 24 h and 58 h post-treatment. The 12 total samples were hybridized against a Cy3-labeled reference pool of 3D7 mixed stage asexual parasites and NF54-pfs16-GFP-LUC mixed stage gametocytes on a two-color array. Early gametocyte samples were harvested from culture by 0.01%(w/v) saponin addition and late-stage samples were harvested via density centrifugation using Nycoprep 1.077 cushions and stored at -80˚C until RNA isolation using TRIZOL reagent along with on-column extraction using the Qiagen RNeasy kit (Qiagen, Germany).