Monitoring <i>Plasmodium falciparum</i> growth and development by UV flow cytometry using an optimized Hoechst‐thiazole orange staining strategy

Grimberg, Brian T.; Erickson, John J.; Sramkoski, R. Michael; Jacobberger, James W.; Zimmerman, Peter A.

doi:10.1002/cyto.a.20541

Cited by 70 publications

(85 citation statements)

References 66 publications

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“…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). In this study, the correlation between numbers of free merozoites and parasite growth inhibition (newly invaded rings) has been described in detail.…”

Section: Discussionmentioning

confidence: 99%

Defining the Timing of Action of Antimalarial Drugs against Plasmodium falciparum

Wilson

Langer

Goodman

et al. 2013

Antimicrob Agents Chemother

131

135

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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.

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Section: Discussionmentioning

confidence: 99%

Defining the Timing of Action of Antimalarial Drugs against Plasmodium falciparum

Wilson

Langer

Goodman

et al. 2013

Antimicrob Agents Chemother

131

135

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“…However, since individual developmental stages generate different amounts of singlet oxygen highly synchronized plasmodial stages are a prerequisite for flow cytometry applications. Alternatively counterstaining with different fluorescent dyes could be applied to enable specific visualization of infected and noninfected erythrocytes as well as different developmental stages of the malaria parasite (24,25).…”

Section: Discussionmentioning

confidence: 99%

Cytometric quantification of singlet oxygen in the human malaria parasitePlasmodium falciparum

et al. 2012

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The malaria parasite Plasmodium falciparum proliferates within human erythrocytes and is thereby exposed to a variety of reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, superoxide anion, and highly reactive singlet oxygen ( 1 O 2 ). While most ROS are already well studied in the malaria parasite, singlet oxygen has been neglected to date. In this study we visualized the generation of 1 O 2 by live cell fluorescence microscopy using 3-(p-aminophenyl) fluorescein as an indicator dye. While 1 O 2 is found restrictively in the parasite, its amount varies during erythrocytic schizogony. Since the photosensitizer cercosporin generates defined amounts of 1 O 2 we have established a new cytometric method that allows the stage specific quantification of 1 O 2 . Therefore, the parasites were first classified into three main stages according to their respective pixel-area of 200-600 pixels for rings, 700-1,200 pixels for trophozoites and 1,400-2,500 pixels for schizonts. Interestingly the highest mean concentration of endogenous 1 O 2 of 0.34 nM is found in the trophozoites stage, followed by 0.20 nM (ring stage) and 0.10 nM (schizont stage) suggesting that 1 O 2 derives predominantly from the digestion of hemoglobin. ' 2012 International Society for Advancement of Cytometry

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“…For RNA and DNA staining (46), rbcs were pelleted and resuspended in 400 μl of Retic-COUNT Reagent (thiazole orange [TO]; BD), and then HO 34580 added at a 4-mM final concentration. A total of 100,000 cells were collected by the LSRFortessa flow cytometer, and data were analyzed by FACSDiva software.…”

Section: Methodsmentioning

confidence: 99%

Cross-species malaria immunity induced by chemically attenuated parasites

Good¹,

Reiman²,

Rodriguez³

et al. 2013

J. Clin. Invest.

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Vaccine development for the blood stages of malaria has focused on the induction of antibodies to parasite surface antigens, most of which are highly polymorphic. An alternate strategy has evolved from observations that low-density infections can induce antibody-independent immunity to different strains. To test this strategy, we treated parasitized red blood cells from the rodent parasite Plasmodium chabaudi with secocyclopropyl pyrrolo indole analogs. These drugs irreversibly alkylate parasite DNA, blocking their ability to replicate. After administration in mice, DNA from the vaccine could be detected in the blood for over 110 days and a single vaccination induced profound immunity to different malaria parasite species. Immunity was mediated by CD4 + T cells and was dependent on the red blood cell membrane remaining intact. The human parasite, Plasmodium falciparum, could also be attenuated by treatment with seco-cyclopropyl pyrrolo indole analogs. These data demonstrate that vaccination with chemically attenuated parasites induces protective immunity and provide a compelling rationale for testing a blood-stage parasite-based vaccine targeting human Plasmodium species.

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Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst‐thiazole orange staining strategy

Cited by 70 publications

References 66 publications

Defining the Timing of Action of Antimalarial Drugs against Plasmodium falciparum

Defining the Timing of Action of Antimalarial Drugs against Plasmodium falciparum

Cytometric quantification of singlet oxygen in the human malaria parasitePlasmodium falciparum

Cross-species malaria immunity induced by chemically attenuated parasites

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