Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition

Jogdand, Prajakta; Singh, S. K.; Christiansen, Michael; Dziegiel, Morten Hanefeld; Singh, Subhash; Theisen, Michael

doi:10.1186/1475-2875-11-235

Cited by 31 publications

(33 citation statements)

References 55 publications

(59 reference statements)

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“…Loss of parasite mitochondrial membrane potential (ΨΔmit) was determined using 5 μM Mitrotracker Red CMXROS as described previously [18]. Cultures were incubated for 30 min at 37°C with the dye and then for 1 h with 10-fold serial dilutions (0.001-100 μM) of N3 and atovaquone.…”

Section: Methodsmentioning

confidence: 99%

Biological evaluation of hydroxynaphthoquinones as anti-malarials

Schuck

Ferreira

Cruz

et al. 2013

Malar J

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BackgroundThe hydroxynaphthoquinones have been extensively investigated over the past 50 years for their anti-malarial activity. One member of this class, atovaquone, is combined with proguanil in Malarone®, an important drug for the treatment and prevention of malaria.MethodsAnti-malarial activity was assessed in vitro for a series of 3-alkyl-2-hydroxy-1,4-naphthoquinones (N1-N5) evaluating the parasitaemia after 48 hours of incubation. Potential cytotoxicity in HEK293T cells was assessed using the MTT assay. Changes in mitochondrial membrane potential of Plasmodium were measured using the fluorescent dye Mitrotracker Red CMXROS.ResultsFour compounds demonstrated IC50s in the mid-micromolar range, and the most active compound, N3, had an IC50 of 443 nM. N3 disrupted mitochondrial membrane potential, and after 1 hour presented an IC50ΔΨmit of 16 μM. In an in vitro cytotoxicity assay using HEK 293T cells N3 demonstrated no cytotoxicity at concentrations up to 16 μM.ConclusionsN3 was a potent inhibitor of mitochondrial electron transport, had nanomolar activity against cultured Plasmodium falciparum and showed minimal cytotoxicity. N3 may serve as a starting point for the design of new hydroxynaphthoquinone anti-malarials.

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Section: Methodsmentioning

confidence: 99%

Biological evaluation of hydroxynaphthoquinones as anti-malarials

Schuck

Ferreira

Cruz

et al. 2013

Malar J

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“…Lastly, while less likely to be an issue due to the clearance of dead parasites by the spleen in vivo, if one was monitoring parasitemia over time with drug treatment or in vitro culture parasitemia over multiple cycles without sub-culture (for example), nonviable parasites clearly identifi able by microscopy would not be distinguished from viable parasites by SYBR Green fl ow cytometry. Dyes measuring mitochondrial membrane potential as a marker for parasite viability have been used for this purpose [ 33 ], albeit measuring the ring stage, which is most prevalent in in vivo samples, is more diffi cult. This source of background emphasizes the need for occasional microscopy spot checks of morphology prior to harvesting certain kinds of assays.…”

Section: Measuring In Vitro Invasion Inhibition By Antibodiesmentioning

confidence: 99%

Measuring Plasmodium falciparum Erythrocyte Invasion Phenotypes Using Flow Cytometry

Bei

Duraisingh

2015

Malaria Vaccines

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Having the ability to rapidly, accurately, and robustly measure Plasmodium falciparum merozoite invasion is a critical component in effective assessment of a blood stage vaccine's mechanism of action. Being able to measure invasion of erythrocytes accurately, objectively and in a high throughput fashion is of critical importance. Here, we describe a simple and robust flow cytometry method that allows for the measurement of the key invasion parameters of parasite multiplication rate and erythrocyte selectivity-both important determinants of disease severity-from the schizont to the ring stage of the parasite's life-cycle, thus separating invasion from growth of the parasite. Importantly, this method is able to accurately detect low levels of parasitemia and heterogeneity within the population that can be missed by enzymatic methods. Lastly, this method has been successfully adapted and employed in field based research settings for parasitemia measurements in vivo, ex vivo, and in vitro and to measure invasion inhibition by antibodies and the use of alternative pathways for invasion.

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“…Merozoites are known to contain a single mitochondrion-like organelle which is reproduced during schizogony (23). The mitochondrial membrane potential is a key indicator of parasite viability that can be revealed using MitoTracker Red FM (MitoT; Invitrogen, Life Technologies), a dye known to stain the intact mitochondrial membrane (24)(25)(26). In some experiments, MitoT was used at a concentration of 200 nM.…”

Section: Schizont Maturation Assessment and Determination Of Ic 50 Smentioning

confidence: 99%

Ex Vivo Maturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites

Chang

Malleret

Russell

et al. 2016

Antimicrob Agents Chemother

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c Ex vivo assay systems provide a powerful approach to studying human malaria parasite biology and to testing antimalarials. For rodent malaria parasites, short-term in vitro culture and ex vivo antimalarial susceptibility assays are relatively cumbersome, relying on in vivo passage for synchronization, since ring-stage parasites are an essential starting material. Here, we describe a new approach based on the enrichment of ring-stage Plasmodium berghei, P. yoelii, and P. vinckei vinckei using a single-step Percoll gradient. Importantly, we demonstrate that the enriched ring-stage parasites develop synchronously regardless of the parasite strain or species used. Using a flow cytometry assay with Hoechst and ethidium or MitoTracker dye, we show that parasite development is easily and rapidly monitored. Finally, we demonstrate that this approach can be used to screen antimalarial drugs.T he spread of artemisinin-resistant malaria parasites in Southeast Asia requires the urgent development of new antimalarial therapeutics (1). Currently, the discovery of drugs with activity against Plasmodium falciparum follows a defined pathway (2, 3). Central to this pathway is quantifying the intrinsic susceptibility of a standard laboratory strain of P. falciparum (usually strain 3D7) to the candidate antimalarial drugs, specifically by defining their half-maximal (50%) inhibitory concentration (IC 50 ) in vitro. Lead antimalarial candidates with the lowest IC 50 s (generally Յ1 M) are then tested against cultures of a range of adapted isolates (often with well-defined resistance phenotypes) or against ex vivo fresh isolates (2,(4)(5)(6). Next, to demonstrate efficacy in vivo, rodent malaria parasite models, in particular, Plasmodium berghei ANKA (7) and P. chabaudi (8) in mice, are used. Unfortunately, it is rare for the IC 50 s of the candidate drugs for rodent malaria parasites to be determined, thus preventing meaningful comparisons of the effects of the candidate drugs on human parasites and rodent malaria parasites.In vitro drug screening methods have been developed for P. berghei ANKA. However, P. berghei ANKA parasite infections are generally asynchronous, and therefore, an in vivo synchronization step is required prior to in vitro culture of the parasite (9). Additionally, rodent malaria parasites have different infection profiles, i.e., different lethalities and synchronicities, which are also influenced by the mouse genetic background and immunity (2). As the in vivo result is confounded by the choice of rodent malaria species and strain, as well as the mouse genetic background, we need a simplified ex vivo assay to accurately determine the intrinsic profile of the susceptibility of murine Plasmodium spp. to antimalarials.In the study described here, we developed a single-step protocol to enrich the ring stages of rodent malaria parasites for shortterm in vitro culture that can be used for in vitro screening and measurement of parasite sensitivities to different antimalarial compounds using flow cytometry. MATERIALS ...

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Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition

Cited by 31 publications

References 55 publications

Biological evaluation of hydroxynaphthoquinones as anti-malarials

Biological evaluation of hydroxynaphthoquinones as anti-malarials

Measuring Plasmodium falciparum Erythrocyte Invasion Phenotypes Using Flow Cytometry

Ex Vivo Maturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites

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