Analysis of malaria parasite‐infected blood by flow cytometry

Jacobberger, James W.; Horan, Paul K.; Hare, J.D.

doi:10.1002/cyto.990040307

Cited by 31 publications

(10 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similar levels of sensitivity were observed in mice infected with P. chabaudi adami DS or P. berghei ANKA. Although mitochondrial membrane potential dyes have previously been employed to assess parasite viability [34,42] and to determine parasitaemia [43,44] to the best of this author’s knowledge the combination of these with DNA specific dyes has not been used to quantify parasitaemia in vivo . In addition to JC-1 and Hoechst, selective, fluorescently labelled antibodies were employed to detect and exclude RBC progenitors and leukocytes, further improving the sensitivity of the assay.…”

Section: Discussionmentioning

confidence: 99%

A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo

Lelliott

Lampkin

McMorran

et al. 2014

Malar J

View full text Add to dashboard Cite

BackgroundMalaria treatments are becoming less effective due to the rapid spread of drug resistant parasites. Increased understanding of the host/parasite interaction is crucial in order to develop treatments that will be less prone to resistance. Parasite invasion of the red blood cell (RBC) is a critical aspect of the parasite life cycle and is, therefore, a promising target for the development of malaria treatments. Assays for analysing parasite invasion in vitro have been developed, but no equivalent assays exist for in vivo studies. This article describes a novel flow cytometric in vivo parasite invasion assay.MethodsExperiments were conducted with mice infected with erythrocytic stages of Plasmodium chabaudi adami strain DS. Exogenously labelled blood cells were transfused into infected mice at schizogony, and collected blood samples stained and analysed using flow cytometry to specifically detect and measure proportions of labelled RBC containing newly invaded parasites. A combination of antibodies (CD45 and CD71) and fluorescent dyes, Hoechst (DNA) and JC-1 (mitochondrial membrane potential), were used to differentiate parasitized RBCs from uninfected cells, RBCs containing Howell-Jolly bodies, leukocytes and RBC progenitors. Blood cells were treated ex vivo with proteases to examine the effects on in vivo parasite invasion.ResultsThe staining and flow cytometry analysis method was accurate in determining the parasitaemia down to 0.013% with the limit of detection at 0.007%. Transfused labelled blood supported normal rates of parasite invasion. Protease-treated red cells resulted in 35% decrease in the rate of parasite invasion within 30 minutes of introduction into the bloodstream of infected mice.ConclusionsThe invasion assay presented here is a versatile method for the study of in vivo red cell invasion efficiency of Plasmodium parasites in mice, and allows direct comparison of invasion in red cells derived from two different populations. The method also serves as an accurate alternative method of estimating blood parasitaemia.

show abstract

Section: Discussionmentioning

confidence: 99%

A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo

Lelliott

Lampkin

McMorran

et al. 2014

Malar J

View full text Add to dashboard Cite

show abstract

“…Development of a rapid flow cytometric test of parasite growth and development has enabled us to observe this dynamic processes during the Pf blood stage infection cycle. Based upon the haploid genome copy number of infected cells (which could be determined by HO fluorescence (56)) and the RNA levels (based on the range of TO fluorescence (42)) within the DNA positive cells, the life cycle stages of the parasite could be identified as proposed (Fig. 1).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2008

Self Cite

View full text Add to dashboard Cite

The complex life cycle of Plasmodium falciparum (Pf) makes it difficult to limit infections and reduce the risk of severe malaria. Improved understanding of Pf blood-stage growth and development would provide new opportunities to evaluate and interfere with successful completion of the parasite's life cycle. Cultured blood stage Pf was incubated with Hoechst 33342 (HO) and thiazole orange (TO) to stain DNA and total nucleic acids, respectively. Correlated HO and TO fluorescence emissions were then measured by flow cytometry. Complex bivariate data patterns were analyzed by manual cluster gating to quantify parasite life cycle stages. The permutations of viable staining with both reagents were tested for optimal detection of parasitized RBC (pRBC). Pf cultures were exposed to HO and TO simultaneously to achieve optimal staining of pRBC and consistent quantification of early and late stages of the replicative cycle (rings through schizonts). Staining of Pf nucleic acids allows for analysis of parasite development in the absence of fixatives, lysis, or radioactivity to enable examination of erythrocytes from parasite invasion through schizont rupture using sensitive and rapid assay procedures. Investigation of the mechanisms by which anti-malarial drugs and antibodies act against different Pf lifecycle stages will be aided by this cytometric strategy. '

show abstract

“…These characteristics make its utilization in malaria drug discovery an important advantage in terms of cost effectiveness and it has already been successfully used for testing human samples (32). However, its application has remained problematic in murine models of malaria due to the presence of a relatively high percentage of erythrocytes that contain significant amounts of nucleic acids (2,6,33). An interesting alternative could be the use of green fluorescent protein-transformed plasmodia, as recently described for P. berghei (34).…”

Section: Discussionmentioning

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

Analysis of malaria parasite‐infected blood by flow cytometry

Cited by 31 publications

References 18 publications

A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo

A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo

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

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

Contact Info

Product

Resources

About