Double staining of <i>Plasmodium falciparum</i> nucleic acids with hydroethidine and thiazole orange for cell cycle stage analysis by flow cytometry

Jouin, H; Daher, Wassim; Khalife, Jamal; Ricard, Isabelle; Puijalon, Odile; Capron, Moníque; Dive, Daniel

doi:10.1002/cyto.a.10110

Cited by 28 publications

(28 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our assay demonstrated three potential markers of drug inhibition: free merozoites, ring stages, and unruptured schizonts, which are readily distinguishable by flow cytometry. In practice, detection of ring stages with non-GFP-fluorescent lines may require the application of an alternative staining method such as the combination of thiazole orange and hydroethidine (44) or Hoechst 33342 and (45). Previous studies have reported an encouraging correlation between flow cytometry and microscopy counts of drug-treated cultures in vitro (45,46) as well as application of flow cytometry to assess drug inhibition of late-stage parasite development (47).…”

Section: Discussionmentioning

confidence: 99%

Defining the Timing of Action of Antimalarial Drugs against Plasmodium falciparum

Wilson

Langer

Goodman

et al. 2013

Antimicrob Agents Chemother

129

135

View full text Add to dashboard Cite

e Most current antimalarials for treatment of clinical Plasmodium falciparum malaria fall into two broad drug families and target the food vacuole of the trophozoite stage. No antimalarials have been shown to target the brief extracellular merozoite form of blood-stage malaria. We studied a panel of 12 drugs, 10 of which have been used extensively clinically, for their invasion, schizont rupture, and growth-inhibitory activity using high-throughput flow cytometry and new approaches for the study of merozoite invasion and early intraerythrocytic development. Not surprisingly, given reported mechanisms of action, none of the drugs inhibited merozoite invasion in vitro. Pretreatment of erythrocytes with drugs suggested that halofantrine, lumefantrine, piperaquine, amodiaquine, and mefloquine diffuse into and remain within the erythrocyte and inhibit downstream growth of parasites. Studying the inhibitory activity of the drugs on intraerythrocytic development, schizont rupture, and reinvasion enabled several different inhibitory phenotypes to be defined. All drugs inhibited parasite replication when added at ring stages, but only artesunate, artemisinin, cycloheximide, and trichostatin A appeared to have substantial activity against ring stages, whereas the other drugs acted later during intraerythrocytic development. When drugs were added to late schizonts, only artemisinin, cycloheximide, and trichostatin A were able to inhibit rupture and subsequent replication. Flow cytometry proved valuable for in vitro assays of antimalarial activity, with the free merozoite population acting as a clear marker for parasite growth inhibition. These studies have important implications for further understanding the mechanisms of action of antimalarials, studying and evaluating drug resistance, and developing new antimalarials.

show abstract

Section: Discussionmentioning

confidence: 99%

Defining the Timing of Action of Antimalarial Drugs against Plasmodium falciparum

Wilson

Langer

Goodman

et al. 2013

Antimicrob Agents Chemother

129

135

View full text Add to dashboard Cite

show abstract

“…To determine the growth rate of parasites, the number of infected red cells was counted over a 10-day period by flow cytometry using a fluorescence-activated cell sorter (FACS) as described previously (19,37), except that hydroethidine (at 50 g/ml) was used to stain infected RBC (18). The starting parasitemia was ϳ0.5% with a 2% hematocrit, and the cultures were diluted every 48 h after counting.…”

Section: Parasites and Transfectionmentioning

confidence: 99%

Systematic Genetic Analysis of the Plasmodium falciparum MSP7-Like Family Reveals Differences in Protein Expression, Location, and Importance in Asexual Growth of the Blood-Stage Parasite

Kadekoppala

Ogun

Howell³

et al. 2010

Eukaryot Cell

View full text Add to dashboard Cite

Proteins located on Plasmodium falciparum merozoites, the invasive form of the parasite's asexual blood stage, are of considerable interest in vaccine research. Merozoite surface protein 7 (MSP7) forms a complex with MSP1 and is encoded by a member of a multigene family located on chromosome 13. The family codes for MSP7 and five MSP7-related proteins (MSRPs). In the present study, we have investigated the expression and the effect of msrp gene deletion at the asexual blood stage. In addition to msp7, msrp2, msrp3, and msrp5 are transcribed, and mRNA was easily detected by hybridization analysis, whereas mRNA for msrp1 and msrp4 could be detected only by reverse transcription (RT)-PCR. Notwithstanding evidence of transcription, antibodies to recombinant MSRPs failed to detect specific proteins, except for antibodies to MSRP2. Sequential proteolytic cleavages of MSRP2 resulted in 28-and 25-kDa forms. However, MSRP2 was absent from merozoites; the 25-kDa MSRP2 protein (MSRP2 25 ) was soluble and secreted upon merozoite egress. The msrp genes were deleted by targeted disruption in the 3D7 line, leading to ablation of full-length transcripts. MSRP deletion mutants had no detectable phenotype, with growth and invasion characteristics comparable to those of the parental parasite; only the deletion of MSP7 led to a detectable growth phenotype. Thus, within this family some of the genes are transcribed at a significant level in asexual blood stages, but the corresponding proteins may or may not be detectable. Interactions of the expressed proteins with the merozoite also differ. These results highlight the potential for unexpected differences of protein expression levels within gene families.The human malaria parasite Plasmodium falciparum continues to be a major public health challenge mainly in developing countries, claiming more than one million lives annually. The invasion of erythrocytes and the subsequent cycles of growth, replication, and rupture of infected cells are responsible for the primary pathological consequences, such as rapid hemolysis consequent upon merozoite release and metabolic acidosis (17). Therefore, the blood stage of the parasite life cycle is a primary target for interventions to combat malaria disease. Merozoite surface proteins (MSPs) are considered to be among the best candidate antigens for inclusion in an antimalarial vaccine (15, 39). Their surface location implicates these proteins in the initial attachment and invasion of red cells by merozoites and offers good accessibility to host antibodies. Several MSPs are being assessed as vaccine candidates (10,12,17,43).Merozoite surface protein 1 (MSP1) is located on the developing merozoite surface in association with at least two other proteins-MSP6 and MSP7. Upon invasion of erythrocytes in culture, this protein complex is shed into the supernatant by proteolytic cleavage (3, 4). The complex is comprised of 4 polypeptides generated by sequential proteolytic cleavage of the MSP1 precursor and one polypeptide each from MSP6 (36-kDa protein [...

show abstract

“…However, they require flow cytometers equipped with ultraviolet lasers that are not available in most laboratories. Nonspecific DNA staining agents, such as thiazole orange (7), acridine orange (8)(9)(10), ethidium bromide (11), propidium iodide (12), or hydroethidine (13)(14)(15), have also been used to stain nucleic acids from infected erythrocytes. Recently, YOYO-1, a cyanine dimer, has been added to this list (16).…”

mentioning

confidence: 99%

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

et al. 2005

View full text Add to dashboard Cite

Background: Microscopic analysis of blood smears is currently the most frequently used method to measure parasitemias in experiments of drug efficacy in murine models of malaria. However, it is subjective and labour intensive, which preclude its utilization in large-scale evaluation programs. Flow cytometry is an alternative method, but due to the limited specificity achieved with the currently available techniques, it has not been widely used in murine models of malaria during preclinical evaluation. We describe a new flow cytometric method based on the differences of autofluorescence and DNA content measured after staining with YOYO-1 that are observed in infected erythrocytes compared with noninfected erythrocytes. Methods: Samples of blood from Plasmodium yoeliiinfected animals were fixed with glutaraldehyde, incubated with RNAase, and stained with YOYO-1 in 96-well plate format. After acquisition, erythrocytes gated in loga-

show abstract

Double staining of Plasmodium falciparum nucleic acids with hydroethidine and thiazole orange for cell cycle stage analysis by flow cytometry

Cited by 28 publications

References 25 publications

Defining the Timing of Action of Antimalarial Drugs against Plasmodium falciparum

Defining the Timing of Action of Antimalarial Drugs against Plasmodium falciparum

Systematic Genetic Analysis of the Plasmodium falciparum MSP7-Like Family Reveals Differences in Protein Expression, Location, and Importance in Asexual Growth of the Blood-Stage Parasite

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

Contact Info

Product

Resources

About