A blood smear was taken at indicated time points and stained with Giemsa (1/10 dilution, VWR, Heverlee, Belgium) to obtain percentages of parasitaemia, reticulocytes and infected reticulocytes. Additionally, 1/500 diluted tail blood was counted using a Bürker chamber to obtain RBC concentrations, from which numbers of iRBCs, reticulocytes and infected reticulocytes per ml were calculated. Images of Giemsa-stained blood smears were taken at a magnification of 100 (100×/1.25 oil objective) with a Leica DM 2000 light microscope equipped with a DFC 295 camera (Leica Bond Max, Leica Microsystems, Diegem, Belgium).
For determination of parasitaemia by flow cytometry [35 (link)], tail blood (10 µl) from infected mice was collected in 1 ml of complete culture medium and cells were fixed in 1 ml of a 0.25% (v/v) glutaraldehyde solution in PBS at 4 °C and kept at 4 °C until analysis. These samples were stained with the DNA-specific fluorescent dye Hoechst-33258 (2 µmol/l) for 1 h at 37 °C and analysed with a a FACScan (LSR II, Becton–Dickinson). The fluorescence intensity and size (Forward Scatter; FSC; Sideward Scatter, SSC) of 50,000 cells per sample were measured and data analysis was performed using the FlowJo software (FlowJo, LLC, Ashland, USA).
For determination of parasitaemia by flow cytometry [35 (link)], tail blood (10 µl) from infected mice was collected in 1 ml of complete culture medium and cells were fixed in 1 ml of a 0.25% (v/v) glutaraldehyde solution in PBS at 4 °C and kept at 4 °C until analysis. These samples were stained with the DNA-specific fluorescent dye Hoechst-33258 (2 µmol/l) for 1 h at 37 °C and analysed with a a FACScan (LSR II, Becton–Dickinson). The fluorescence intensity and size (Forward Scatter; FSC; Sideward Scatter, SSC) of 50,000 cells per sample were measured and data analysis was performed using the FlowJo software (FlowJo, LLC, Ashland, USA).
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