Plasmodium species: Flow cytometry and microfluorometry assessments of DNA content and synthesis

Abstract

. 1987. Plasmodium species: Flow cytometry and microfluorometry assessments of DNA content and synthesis. Experimental Parasitology 63, 88-94. Fluorescence intensities were established by flow cytometry of different erythrocytic stages of Plasmodium berghei after staining of their DNA with Hoechst-33258 or Hoechst-33342. Parasites were obtained from highly synchronized infections or in vitro cultures. Most fluorescence measurements were performed using a low cost, clinical flow cytometer, equipped with a mercury arc lamp. Cells infected with P. berghei could be readily distinguished from uninfected cells on the basis of Hoechst-DNA fluorescence and single, double, and triple ring infected cells were separated clearly. The relative fluorescence intensities of different developmental stages (merozoites, ringforms, trophozoites, schizonts, and gametocytes) corresponded closely to the relative DNA contents of these stages as measured by microfluorometry. Flow cytometry appeared to be a sensitive and rapid method to measure DNA synthesis during asexual development; a C, value of 5 p,&f of aphidicolin, a specific inhibitor of DNA synthesis, was established. Vital staining of parasites in culture was possible with both Hoechst dyes. After removal of Hoechst-33258, normal in vitro development of the stained parasites was observed. After Hoechst staining, the haploid ringforms of P. vivax showed slightly less fluorescence (15%) than ringforms of P. berghei and P. falciparum. No differences in fluorescence intensity were observed, however, by direct microfluorometry after Feulgen-pararosaniline staining, indicating that all three species have the same DNA content. 6

Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

“…FCA have proved to be useful to study diverse features related with human and experimental malaria (Janse et al 1987, Pattanapanyasat et al 1993, 1999, Kumaratilake & Ferrante 2000, Saito-Ito et al 2001. In this study, we have described by flow cytometry the anti-malarial activity of four drugs using continuos cultures of P. falciparum geographically different strains.…”

Improvement of detection specificity of Plasmodium‐infected murine erythrocytes by flow cytometry using autofluorescence and YOYO‐1

“…Because infected erythrocytes contain nucleic acids from the different stages of Plasmodium, the former can be easily identified and counted by flow cytometry using DNA staining dyes. Among these, Hoescht 33258 and Hoescht 33342 are able to cross cellular membranes and are specific for DNA (3)(4)(5)(6). However, they require flow cytometers equipped with ultraviolet lasers that are not available in most laboratories.…”

Evaluation of Purine Salvage as a Chemotherapeutic Target in the Plasmodium yoelli Rodent Model

“…It is possible that flow cytometry may not be able to detect all the infected from the uninfected cells based on GFP-fluorescence intensity, thus leading to an underestimate of the percentage of GFP-positive infected erythrocytes. Co-labeling with the DNA-specific dye Hoechst 33258, which will improve detection of infected erythrocytes, can enhance the sensitivity of this approach [12,13]. Furthermore, this may be compensated for by enriching for more mature stages prior to sorting, or adjusting for the percentage of rings versus more mature stages in the sample being analyzed.…”