Figure 1. Rosetting in static and flow conditions.

A) Parasite strain IT/R29 under static conditions. IEs at 5% parasitaemia and 1% haematocrit were stained with 25 µgml ethidium bromide to visualize the parasite DNA, seen as bright white areas within IEs. Wet preparations were viewed under dual brightfield/UV fluorescence (×100). The images were captured using a Q-imaging camera attached to an Olympus BX50 microscope. B) Parasite strain IT/R29 under flow. 3 ml of parasite culture at 5% parasitaemia and 1% haematocrit was flowed over a µ-slide I (ibidi, Germany) at 0.5 dyn/cm² for 5 minutes. The flow was stopped and images captured immediately using an EVOS FL inverted fluorescence microscope (×20). IEs were stained with 1∶10,000 dilution of SyBr Green prior to the assay to identify IEs within the rosettes clearly. White arrows indicate “multi-rosettes” seen under flow conditions. C) Parasite strain TM284R+ under static conditions, as described in A. D) Parasite strain TM284R+ under flow as described in B. E) IT/R29 multi-rosette from a flow experiment at 0.5 dyn/cm², demonstrating the use of ImageJ to draw the circumference and measure the surface area of the selected multi-rosette and expressed as µm².