Fig 1. Representative traces illustrating the strategy employed to screen the Malaria Box for compounds that perturb the cytosolic pH (pH_cyt) of the parasite.

Malaria Box compounds (1 μM; from the May 2012 Malaria Box batch; identified here by their positions on the plates) were added successively to BCECF-loaded isolated 3D7 P. falciparum trophozoites (suspended at 37°C in pH 7.10 Experimental Saline Solution). A maximum of nine inactive compounds were tested on a single batch of parasites before the V-type H⁺-ATPase inhibitor concanamycin A (conA; 100 nM) was added as a positive control to ensure that a pH change was still detectable (A). A batch of parasites was also replaced with a new batch after a hit was detected (B) or a compound caused an optical effect (seen as an instantaneous perturbation of the fluorescence signal; C). In the latter case concanamycin A (100 nM) was added before changing to another batch of parasites. The purpose of adding the concanamycin A was to confirm (or otherwise) that any such optical effect did not obscure the pH-responsiveness of the fluorescence signal.