A simple monochromatic flow cytometric assay for assessment of intraerythrocytic development of Plasmodium falciparum

Abstract

Background: Gold standard microscopic examination of Plasmodium falciparum intraerythrocytic stage remains an important process for staging and enumerating parasitized erythrocytes in culture; however, microscopy is laborious and its accuracy is dependent upon the skill of the examiner. Methods:In this study, ViSafe Green (VSG), which is a nucleic acid-binding fluorescent dye, was used for assessing in vitro development of P. falciparum using flow cytometry.Results: Fluorescence intensity of VSG was found to depend on the developmental stage of parasites. Specifically, multiple-nuclei-containing schizonts were observed in the VSG high population, and growing trophozoites and ringshaped forms were observed in the VSG intermediate and VSG low populations. The efficacy of VSG-based assay was found to be comparable to the microscopic examination method, and it demonstrated an ability to detect as low as 0.001% of the parasitaemia estimated by Giemsa staining. Moreover, when applying VSG for anti-malarial drug test, it was able to observe the growth inhibitory effect of dihydroartemisinin, the front-line drug for malaria therapy. Conclusions:Taken together, the results of this study suggest the VSG-based flow cytometric assay to be a simple and reliable assay for assessing P. falciparum malaria development in vitro.

Fluorescent Nitrogen-Doped Carbon Dots for Label Live Elder Blood-Stage Plasmodium falciparum through New Permeability Pathways

“…TO is a cyanine stain which is based on thioflavin T and it stains primarily RNA [24]. Both stainings, HE and TO, had high efficiency to detect parasites at the blood stage as described in previous study and were confirmed again in this study [26,27].…”

Trio fluorophore-based phenotypic assay for the detection of artemisinin-induced growth-arrested Plasmodium falciparum in human erythrocytes

“…To prove the concept, we selected cell-permeant and cell-impermeant fluorophores that emit maximum fluorescence signals at different wavelengths and are applicable for most flow cytometers equipped with common lasers. Previously, our group reported the use of the cell-permeant ViSafe green (VSG) to detect P. falciparum -infected erythrocytes 21 . Given its common use in cell survival tests, the cell-impermeant PI was selected to combine with VSG.…”

“…ViSafe Green (VSG) is a cell-permeable fluorophore that was originally used for visualizing separated DNA or RNA on agarose or polyacrylamide gels. Our group demonstrated that VSG could be used to assess the development of P. falciparum in vitro 21 . However, one significant disadvantage of single-use DNA fluorochromes is that they cannot differentiate between viable and dead parasites.…”

Molecular identification of native Wolbachia pipientis in Anopheles minimus in a low-malaria transmission area of Umphang Valley along the Thailand-Myanmar border

“…The gDNA of Mansonia spp. or Plasmodium falciparum strain K1 [ 42 ] were used as positive control, respectively. Nuclease-free water was set as the no template control.…”