Fig. 3From: The effects of dyslipidaemia and cholesterol modulation on erythrocyte susceptibility to malaria parasite infectionWorkflow 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]Back to article page