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

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+.

细胞内钾离子（K+）的测量可通过离子选择性电极、X射线显微分析、高效液相色谱法、火焰光度法和原子吸收光谱法进行。然而，这些技术往往需要复杂的样品制备，并且通常需要专用设备。通过采用基于荧光的整细胞分析技术，该技术不会破坏样品，且可使用诸如荧光计或流式细胞仪等易于获得的设备，可以克服上述限制。在本研究中，我们开发了互补的荧光分析方法，使用膜电位染料DiBAC4(3)和钾离子敏感指示剂APG-1，分别评估膜电位（Δψ）和细胞内钾离子的变化。我们发现，250 nM的DiBAC4(3)在30分钟孵育后，在分离的无性疟原虫滋养体中提供了高荧光信号，该条件产生了信号与噪声比为119.28和Z’-因子为0.83，并用于进一步分析在化合物处理后寄生虫膜电位的改变。5 μM的APG-1在60分钟后产生了最高的信号与背景比，同时也产生了高信号与噪声比（276.26）和Z’-因子为0.89。因此，这两种荧光探针可以在疟原虫寄生虫中成功检测，并随后评估Δψ和细胞内钾离子的变化。