Two-color flow-cytometric analysis of the growth cycle of Plasmodium falciparum in vitro: identification of cell cycle compartments.

Abstract

A previous study (Hare JD, Bahler DW: J Histochem Cytochem 34:215, 1986) has shown that the flow cytometric analysis of acridine-orange-stained Plasmodium falciparum growing in vitro generates a complex two-color display, regions of which correlate with the major morphological stages. In this report, four cell cycle compartments (A-D) are defined by characteristic ratios of red and green fluorescence of cells distributed throughout the erythrocytic cycle as well as by the differential effects of several metabolic inhibitors. The primary characteristic of cells in compartment A is the significant increase in red fluorescence. Inhibition of DNA synthesis by either aphidicolin or hydroxyurea causes the accumulation of cells at the interface between compartments A and B, whereas n-butyrate prevents cells in compartment A from reaching the A-B interface. Cells in compartment A display a small increase in green fluorescence which is independent of DNA synthesis but is enhanced by n-butyrate treatment. Cells in compartment B display a continued increase in red fluorescence coupled with a significant increase in green fluorescence, reflecting the onset of DNA synthesis in compartment B. The transition to compartment C is more abrupt and is associated with a marked increase in green fluorescence and little increase in red fluorescence. Compartment D is characterized by an increase in red fluorescence and a continued rise in green fluorescence. It is postulated that these discontinuities in the two-color display reflect not only changes in the rates of RNA and DNA synthesis but also decondensation of parasite chromatin in compartment A as the organism prepares for DNA synthesis, and re-condensation in compartment D as the newly replicated chromatin prepares for segregation into merozoites. The method described promises to provide a sensitive and rapid technique to study the effects of various factors on the growth cycle of the parasite.

Methodology and application of flow cytometry for investigation of human malaria parasites

“…Several groups have tried to infer the life cycle stage of the malaria parasite based on their cytometry data resulting from AO staining (Hare, 1986; Hare and Bahler, 1986; Shapiro and Mandy, 2007). Theoretically, this is possible because the ring stages generally do not express much RNA and have no DNA synthesis so they should fluoresce exclusively green.…”

Isolation of Plasmodium falciparum by flow-cytometry: implications for single-trophozoite genotyping and parasite DNA purification for whole-genome high-throughput sequencing of archival samples

“…In the case of malaria research, methods of sorting have already been developed to specifically deal with these kinds of samples (cultivated or short terms cultivated parasites) [9,14,24,25]. These methods could, in principle, also be adapted to perform single cell genotyping.…”

Light depolarization measurements in malaria: A new job for an old friend

“…Cytometry of malaria-infected RBC has been used for (i) monitoring of infected RBC, mainly in mouse models (34)(35)(36)(37)(38)(39), (ii) characterization of infected RBC (15), (iii) assessment of maturation and/or viability of parasites in infected RBC (10,13,14,16,17,40,41), and (iv) to detect and count P. falciparum infected RBC in culture (14,(42)(43)(44) or from patient blood (45)(46)(47), with the possibility to distinguish species based on base composition (2,10,11).…”

“…By 1987, it had been shown that flow cytometry of DNA and RNA content of iRBCs could differentiate stages and, in some cases, species (10,11). Flow cytometry has since been used to study parasite physiology and response to antimalarial drugs (12)(13)(14)(15)(16)(17).…”