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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.

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Related in: MedlinePlus

Confocal visualization of engulfed erythrocytes in monocytes and macrophages.Incubation of erythrocytes with phagocytes was carried out for 4 h at 37°C with an E:T ratio of 1∶100 and the phagocytes are subsequently labeled with FITC anti-human CD36 before viewing under the confocal microscope. Z-stack sections of A) a monocyte containing a CellTrace™ Violet- labeled uRBC or an iRBC labeled with both CellTrace™ Violet and DHE, B) a macrophage containing a CellTrace™ Violet- labeled uRBC or an iRBC labeled with both CellTrace™ Violet and DHE. (The scale bar represents 5 µm).
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pone-0038523-g006: Confocal visualization of engulfed erythrocytes in monocytes and macrophages.Incubation of erythrocytes with phagocytes was carried out for 4 h at 37°C with an E:T ratio of 1∶100 and the phagocytes are subsequently labeled with FITC anti-human CD36 before viewing under the confocal microscope. Z-stack sections of A) a monocyte containing a CellTrace™ Violet- labeled uRBC or an iRBC labeled with both CellTrace™ Violet and DHE, B) a macrophage containing a CellTrace™ Violet- labeled uRBC or an iRBC labeled with both CellTrace™ Violet and DHE. (The scale bar represents 5 µm).

Mentions: It was also interesting to note that a large proportion of the increase in phagocytosis observed for P. falciparum-infected cultures in the various experiments was attributed to uptake of uRBCs and not just iRBCs (Figure 4, 5), especially with monocytes. The monocytes showed a high uRBC uptake from infected cultures compared to the macrophages, particularly after P. falciparum (+) serum opsonisation. Samples were observed under confocal microscopy. Figure 6 shows 3D z-stack sections of phagocytes which have ingested either uRBCs or iRBCs from P. falciparum-infected cultures. FITC anti-human CD36 labeling the phagocyte surface demonstrated the erythrocytes were indeed ingested by the phagocytes. As expected, the level of FITC fluorescence was lower in monocytes than in macrophages, due to the abundant expression of CD36 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)

Confocal visualization of engulfed erythrocytes in monocytes and macrophages.Incubation of erythrocytes with phagocytes was carried out for 4 h at 37°C with an E:T ratio of 1∶100 and the phagocytes are subsequently labeled with FITC anti-human CD36 before viewing under the confocal microscope. Z-stack sections of A) a monocyte containing a CellTrace™ Violet- labeled uRBC or an iRBC labeled with both CellTrace™ Violet and DHE, B) a macrophage containing a CellTrace™ Violet- labeled uRBC or an iRBC labeled with both CellTrace™ Violet and DHE. (The scale bar represents 5 µm).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038523-g006: Confocal visualization of engulfed erythrocytes in monocytes and macrophages.Incubation of erythrocytes with phagocytes was carried out for 4 h at 37°C with an E:T ratio of 1∶100 and the phagocytes are subsequently labeled with FITC anti-human CD36 before viewing under the confocal microscope. Z-stack sections of A) a monocyte containing a CellTrace™ Violet- labeled uRBC or an iRBC labeled with both CellTrace™ Violet and DHE, B) a macrophage containing a CellTrace™ Violet- labeled uRBC or an iRBC labeled with both CellTrace™ Violet and DHE. (The scale bar represents 5 µm).
Mentions: It was also interesting to note that a large proportion of the increase in phagocytosis observed for P. falciparum-infected cultures in the various experiments was attributed to uptake of uRBCs and not just iRBCs (Figure 4, 5), especially with monocytes. The monocytes showed a high uRBC uptake from infected cultures compared to the macrophages, particularly after P. falciparum (+) serum opsonisation. Samples were observed under confocal microscopy. Figure 6 shows 3D z-stack sections of phagocytes which have ingested either uRBCs or iRBCs from P. falciparum-infected cultures. FITC anti-human CD36 labeling the phagocyte surface demonstrated the erythrocytes were indeed ingested by the phagocytes. As expected, the level of FITC fluorescence was lower in monocytes than in macrophages, due to the abundant expression of CD36 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