Cellquest pro software 6.0.4

The levels of VAR2CSA present was determined using flow cytometry, as previously described71 (link). In short, infected erythrocytes at trophozoite stages (24–26 h post infection) were taken and washed with PBS supplemented with 2% fetal calf serum (FCS). A rabbit antiserum against baculovirus-produced recombinant domains of VAR2CSA was used. A volume of 3 μl of sera was used to label the samples for 30 min in a final volume of 50 μl PBS/FCS. After several washing steps, the samples were then labelled with 1:100 Alexa 488 goat anti-rabbit IgG (H+L) (Life Technologies) and 1:100 propidium iodide for 30 min in a final volume of 50 ml PBS/FCS. Uninfected erythrocytes were treated concurrently throughout as control. The fluorescence was finally determined using a FACScalibur (Becton Dickinson) and the CellQuest Pro Software 6.0.4 BD (Franklin Lakes).

The presentation of total antigens was monitored using flow cytometry. Erythrocytes were taken from the culture at specific time points and fixed with 0.05% glutaraldehyde for 15 min. Afterwards, the cells were washed with 2% fetal calf serum in PBS. The cells were then labeled for 30 min with 3 µl of either pooled immune serum from 2 adult individuals living in a hyperendemic region in Burkina Faso or one uninfected adult as a control in a final volume of 50 µl PBS/fetal calf serum. After multiple washing steps, the erythrocytes were resuspended in 50 µl PBS/fetal calf serum containing 1∶100 fluorescein (FITC)-conjugated affinity-pure F(ab′)2 fragment donkey anti-human IgG (H+L), (Jackson ImmunoResearch Laboratories) and 1∶100 propidium iodide for 30 min. Uninfected erythrocytes were similarly treated in parallel as a control. After further washing steps, the fluorescence signal was measured. Briefly, the fluorescence displayed on the surface of 400 infected erythrocytes was determined using a FACScalibur (Becton Dickinson) and the CellQuest Pro Software 6.0.4 BD (Franklin Lakes) was used to further process and analyze the data.