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Fig. 3 | Malaria Journal

Fig. 3

From: The effects of dyslipidaemia and cholesterol modulation on erythrocyte susceptibility to malaria parasite infection

Fig. 3

Workflow of the Plasmodium berghei merozoite assay. Firstly, the blood from the mouse (or mice) of interest is harvested the day prior to the invasion assay either by tail-bleed or cardiac puncture. For the assay to be quantitative, the number of cells need to be kept equal between samples obtained from different mice. This is done using a flow cytometric counting assay. Secondly, GFP-positive P. berghei are injected and grown in mice. At the appropriate parasitaemia, the blood is harvested by cardiac puncture 1 day prior to the invasion assay and incubated at 37 °C under low oxygen conditions until the parasites have developed into mature schizonts (12–24 h). The mature parasites are then separated from uninfected blood using a MACS magnetic cell separator, and passed through a 1.2 micrometre filter to rupture the parasitophorous vacuolar membrane and release individual merozoites [22]. Next, in a 96 well plate, three wells per mouse, each with 1 × 106 blood cells, are set up before a fixed volume of free merozoites is added to each well. Invasion is allowed to occur for 30 min at 37 °C while shaking. Finally, the samples are stained with EtBr and analysed by flow cytometry. The population of erythrocytes invaded by parasites is gated on based on their GFP-positive, low EtBr staining, as the dye does not stain the intra-erythrocytic parasite as efficiently [28]

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