Figure 1.
Ring-staged 3D7 Plasmodium falciparum culture labeled with varying concentrations of dihydroethidium.
Ring-staged cultures at about 15% parasitemia, 1% hematocrit were labeled with A) 5 µg/ml, B) 10 µg/ml, C) 25 µg/ml and D) 50 µg/ml DHE for 20 min at 37°C and each was compared to an (gray shade histogram) unstained blood control using flow cytometry. Cultures labeled with 5 µg/ml DHE showed a clear resolution between infected and uninfected erythrocytes (obtaining a parasitemia of 14.42% which corresponded to that in the Giemsa blood smear) and a low uRBC background as compared to the other concentrations.
Figure 2.
Comparison between EB and DHE labeling, using Hoechst 33342 as a comparator.
Ring-staged Plasmodium falciparum cultures at 20% parasitemia, 1% hematocrit were labeled with (A, C) 10 µg/ml EB or (B, D) 5 µg/ml DHE, both of which were co-stained with 1 µg/ml Hoechst 33342, for 20 min at 37°C before analysis using flow cytometry and confocal imaging. A) Only about 20% of Hoechst-labeled iRBCs were stained with EB whereas B) more than 90% of Hoechst-labeled iRBCs were stained with DHE.
Figure 3.
Optimisation of effector (THP-1) to target (erythrocytes) ratio.
The effectors used in these experiments were (A) THP-1 monocytes, (B) PMA-differentiated THP-1 macrophages and the targets were fresh uninfected erythrocytes (uRBC) or ring-staged Plasmodium falciparum culture (ring culture) at 10% parasitemia. ____Percentage of effectors that have engulfed at least one erythrocyte (uninfected or infected) at effector : target (E:T) ratios from 1∶10 to 1∶260 after 4 h incubation at 37°C with pure effectors as the control. The experiments were done in duplicates at least 3 times with data expressed as mean ± SEM. Statistical analyses compared the number of effectors which had ingested erythrocytes from the uRBC culture with that from ring culture at the same E:T ratio. (*represents p<0.05, **represent p<0.005, ***represent p<0.001).
Figure 4.
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 monocytes.
In standard conditions, incubation of erythrocytes with monocytes was carried out for 4 h at 37°C with an E:T ratio of 1∶100. To prevent phagocytosis, monocytes 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 monocytes. A) Monocytes that have engulfed at least one uRBC and B) monocytes that have engulfed at least one iRBC. Data expressed as mean ± SEM. (B*vE*, p<0.05; a*vd*, p<0.005; A*vF*, C*vD*, F*vI*, G*vH*, b*vc*, f*vg*, p<0.001; e*vh*, p = 0.063; A*vB*, p = 0.097; n = 3 separate experiments, each in duplicates) C) Representative dotplots of the respective conditions when exposed to THP-1 monocytes.
Figure 5.
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.
Figure 6.
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).