Limits...
A simplified, sensitive phagocytic assay for malaria cultures facilitated by flow cytometry of differentially-stained cell populations.

Chan CL, Rénia L, Tan KS - PLoS ONE (2012)

Bottom Line: The sensitivity of the method was tested using varying conditions including phagocyte type (monocytes versus macrophages), parasite stage (rings versus schizonts), and negative (incubation with cytochalasin D) and positive (incubation with immune sera) effectors of phagocytosis.It does away with the need to purify infected cells, allowing the study of effects on neighboring uninfected cells.This method may also be translated for use with different types of phagocytes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cellular Parasitology, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT

Background: Phagocytosis of infected and uninfected erythrocytes is an important feature of malaria infections. Flow cytometry is a useful tool for studying phagocytic uptake of malaria-infected erythrocytes in vitro. However, current approaches are limited by the inability to discriminate between infected and uninfected erythrocytes and a failure to stain the early developmental ring stages of infected erythrocytes. The majority of infected erythrocytes in circulation are of the ring stage and these are therefore important targets to study.

Methodology/principal findings: In vitro P. falciparum cultures comprising infected and uninfected erythrocytes were labeled and exposed to cells derived from the human monocytic THP-1 cell line. Phagocytosis was assayed by flow cytometry. Dual labeling of Plasmodium DNA and erythrocyte cytoplasm with dihydroethidium and CellTrace™ Violet respectively allowed, for the first time, the detection and enumeration of phagocytes with ingested erythrocytes from both early ring- and late schizont-stage P, falciparum cultures. The sensitivity of the method was tested using varying conditions including phagocyte type (monocytes versus macrophages), parasite stage (rings versus schizonts), and negative (incubation with cytochalasin D) and positive (incubation with immune sera) effectors of phagocytosis. The current assay clearly demonstrated uptake of infected and uninfected erythrocytes exposed to phagocytes; the extent of which was dependent on the conditions mentioned.

Conclusions: We describe a simple, sensitive and rapid method for quantifying phagocytosis of P. falciparum-infected erythrocytes, by flow cytometry. This approach can be applied for studying parasite-phagocyte interactions under a variety of conditions. The investigation of phagocytosis of P. falciparum-infected erythrocytes can extend from looking solely at late-staged infected erythrocytes to include early-staged ones as well. It does away with the need to purify infected cells, allowing the study of effects on neighboring uninfected cells. This method may also be translated for use with different types of phagocytes.

Show MeSH

Related in: MedlinePlus

Phagocytosis of fresh uninfected erythrocytes (uRBC), ring-staged (ring culture) and schizont-staged cultures (schizont culture) at 10% parasitemia under different conditions by THP-1 differentiated macrophages.In standard conditions, incubation of erythrocytes with macrophages was carried out for 4 h at 37°C with an E:T ratio of 1∶100. To prevent phagocytosis, macrophages were pretreated with 5 µM cytochalasin D for 1 h before the phagocytic incubation of 4 h at 37°C. To increase phagocytosis, erythrocytes were opsonised with serum from a Plasmodium falciparum (+) patient at room temperature for 30 min before incubation with macrophages. A) Macrophages that have engulfed at least one uRBC and B) macrophages that have engulfed at least one iRBC. Data expressed as mean ± SEM. (bvc, p<0.05; BvE, p<0.005; AvF, CvD, FvI, GvH, evh, fvg, p<0.001; avd, p = 0.098; n = 3 separate experiments, each in duplicates) C) Representative dotplots of the respective conditions when exposed to THP-1 macrophages.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3366917&req=5

pone-0038523-g005: Phagocytosis of fresh uninfected erythrocytes (uRBC), ring-staged (ring culture) and schizont-staged cultures (schizont culture) at 10% parasitemia under different conditions by THP-1 differentiated macrophages.In standard conditions, incubation of erythrocytes with macrophages was carried out for 4 h at 37°C with an E:T ratio of 1∶100. To prevent phagocytosis, macrophages were pretreated with 5 µM cytochalasin D for 1 h before the phagocytic incubation of 4 h at 37°C. To increase phagocytosis, erythrocytes were opsonised with serum from a Plasmodium falciparum (+) patient at room temperature for 30 min before incubation with macrophages. A) Macrophages that have engulfed at least one uRBC and B) macrophages that have engulfed at least one iRBC. Data expressed as mean ± SEM. (bvc, p<0.05; BvE, p<0.005; AvF, CvD, FvI, GvH, evh, fvg, p<0.001; avd, p = 0.098; n = 3 separate experiments, each in duplicates) C) Representative dotplots of the respective conditions when exposed to THP-1 macrophages.

Mentions: After incubation with phagocytes for 4 h, there was a significant increase in the uptake of uRBC in schizont cultures compared to fresh uninfected cultures. However, a similar comparison between uRBC in ring cultures and fresh uninfected cultures was not significant in both monocytes and macrophages. With monocytes, uptake of fresh uRBC was 3.5±0.4% compared to uRBC from schizont cultures (10.4±1.6%; p<0.001; Figure 4A). Fresh uRBC uptake by macrophages was 1.9±0.4% compared to uRBC from schizont cultures (5.6±1.1%; p<0.001; Figure 5A). We also noticed an increase in macrophage phagocytosis of schizont-staged iRBC (4.4±0.2%) compared to ring-staged iRBC (1.6±0.3%) with p<0.005 (Figure 5B) but not in monocytes (Figure 4B). The uptake of ring and schizont iRBCs in monocytes were similar to ring iRBC uptake in macrophages.


A simplified, sensitive phagocytic assay for malaria cultures facilitated by flow cytometry of differentially-stained cell populations.

Chan CL, Rénia L, Tan KS - PLoS ONE (2012)

Phagocytosis of fresh uninfected erythrocytes (uRBC), ring-staged (ring culture) and schizont-staged cultures (schizont culture) at 10% parasitemia under different conditions by THP-1 differentiated macrophages.In standard conditions, incubation of erythrocytes with macrophages was carried out for 4 h at 37°C with an E:T ratio of 1∶100. To prevent phagocytosis, macrophages were pretreated with 5 µM cytochalasin D for 1 h before the phagocytic incubation of 4 h at 37°C. To increase phagocytosis, erythrocytes were opsonised with serum from a Plasmodium falciparum (+) patient at room temperature for 30 min before incubation with macrophages. A) Macrophages that have engulfed at least one uRBC and B) macrophages that have engulfed at least one iRBC. Data expressed as mean ± SEM. (bvc, p<0.05; BvE, p<0.005; AvF, CvD, FvI, GvH, evh, fvg, p<0.001; avd, p = 0.098; n = 3 separate experiments, each in duplicates) C) Representative dotplots of the respective conditions when exposed to THP-1 macrophages.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366917&req=5

pone-0038523-g005: Phagocytosis of fresh uninfected erythrocytes (uRBC), ring-staged (ring culture) and schizont-staged cultures (schizont culture) at 10% parasitemia under different conditions by THP-1 differentiated macrophages.In standard conditions, incubation of erythrocytes with macrophages was carried out for 4 h at 37°C with an E:T ratio of 1∶100. To prevent phagocytosis, macrophages were pretreated with 5 µM cytochalasin D for 1 h before the phagocytic incubation of 4 h at 37°C. To increase phagocytosis, erythrocytes were opsonised with serum from a Plasmodium falciparum (+) patient at room temperature for 30 min before incubation with macrophages. A) Macrophages that have engulfed at least one uRBC and B) macrophages that have engulfed at least one iRBC. Data expressed as mean ± SEM. (bvc, p<0.05; BvE, p<0.005; AvF, CvD, FvI, GvH, evh, fvg, p<0.001; avd, p = 0.098; n = 3 separate experiments, each in duplicates) C) Representative dotplots of the respective conditions when exposed to THP-1 macrophages.
Mentions: After incubation with phagocytes for 4 h, there was a significant increase in the uptake of uRBC in schizont cultures compared to fresh uninfected cultures. However, a similar comparison between uRBC in ring cultures and fresh uninfected cultures was not significant in both monocytes and macrophages. With monocytes, uptake of fresh uRBC was 3.5±0.4% compared to uRBC from schizont cultures (10.4±1.6%; p<0.001; Figure 4A). Fresh uRBC uptake by macrophages was 1.9±0.4% compared to uRBC from schizont cultures (5.6±1.1%; p<0.001; Figure 5A). We also noticed an increase in macrophage phagocytosis of schizont-staged iRBC (4.4±0.2%) compared to ring-staged iRBC (1.6±0.3%) with p<0.005 (Figure 5B) but not in monocytes (Figure 4B). The uptake of ring and schizont iRBCs in monocytes were similar to ring iRBC uptake in macrophages.

Bottom Line: The sensitivity of the method was tested using varying conditions including phagocyte type (monocytes versus macrophages), parasite stage (rings versus schizonts), and negative (incubation with cytochalasin D) and positive (incubation with immune sera) effectors of phagocytosis.It does away with the need to purify infected cells, allowing the study of effects on neighboring uninfected cells.This method may also be translated for use with different types of phagocytes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cellular Parasitology, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT

Background: Phagocytosis of infected and uninfected erythrocytes is an important feature of malaria infections. Flow cytometry is a useful tool for studying phagocytic uptake of malaria-infected erythrocytes in vitro. However, current approaches are limited by the inability to discriminate between infected and uninfected erythrocytes and a failure to stain the early developmental ring stages of infected erythrocytes. The majority of infected erythrocytes in circulation are of the ring stage and these are therefore important targets to study.

Methodology/principal findings: In vitro P. falciparum cultures comprising infected and uninfected erythrocytes were labeled and exposed to cells derived from the human monocytic THP-1 cell line. Phagocytosis was assayed by flow cytometry. Dual labeling of Plasmodium DNA and erythrocyte cytoplasm with dihydroethidium and CellTrace™ Violet respectively allowed, for the first time, the detection and enumeration of phagocytes with ingested erythrocytes from both early ring- and late schizont-stage P, falciparum cultures. The sensitivity of the method was tested using varying conditions including phagocyte type (monocytes versus macrophages), parasite stage (rings versus schizonts), and negative (incubation with cytochalasin D) and positive (incubation with immune sera) effectors of phagocytosis. The current assay clearly demonstrated uptake of infected and uninfected erythrocytes exposed to phagocytes; the extent of which was dependent on the conditions mentioned.

Conclusions: We describe a simple, sensitive and rapid method for quantifying phagocytosis of P. falciparum-infected erythrocytes, by flow cytometry. This approach can be applied for studying parasite-phagocyte interactions under a variety of conditions. The investigation of phagocytosis of P. falciparum-infected erythrocytes can extend from looking solely at late-staged infected erythrocytes to include early-staged ones as well. It does away with the need to purify infected cells, allowing the study of effects on neighboring uninfected cells. This method may also be translated for use with different types of phagocytes.

Show MeSH
Related in: MedlinePlus