University of Pretoria
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Fluorescence-based assays to detect changes in membrane potential and K+ levels in isolated Plasmodium falciparum trophozoites

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Intracellular K+ can be measured using ion-selective electrodes, X-ray microanalysis, HPLC, flame photometry and atomic absorption. However, these techniques require complex sample preparation and often specialized equipment. These limitations can be overcome by using a fluorescent-based whole-cell assay that does not destroy the sample and use readily available equipment such as fluorimeters or flow cytometers. Here, we developed complementary fluorescent assays using the membrane potential dye DiBAC4(3) and the K+ sensitive indicator APG-1 to evaluate changes in membrane potential (Δψ) and intracellular K+, respectively.

We found that 250 nM DiBAC4(3) provided a high fluorescent signal in isolated asexual P. falciparum trophozoites after 30 min incubation. This condition resulted in a signal-to-noise of 119.28 and a Z’-factor of 0.83 and was used for further analysis of changes in the parasite’s membrane potential after treatment with compounds. 5 μM APG-1 resulted in the highest signal-to-background ratio after 60 minutes, that also resulted in high signal-to-noise ratios (276.26) and a Z’-factor of 0.89. Therefore, the two fluorescent probes could successfully be detected in P. falciparum parasites and subsequently evaluate changes in Δψ and intracellular K+.

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Biochemistry, Genetics and Microbiology

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