Figure 4. The effect of heparin on rosetting in static and flow assays with parasite strain IT/R29.

Heparin was added to IT/R29 parasite culture suspension at a final concentration of 100, 10 and 1 µg/ml and incubated for 30 mins before assessment of rosetting. The control was a culture suspension with no added heparin. A) Static assay: the percentage in IEs in rosettes (rosette frequency) was assessed by fluorescence microscopy of ethidium bromide-stained wet preparations. B–D) Flow assays: total rosette area (µm²) from 10 fields (20× objective) was determined after 5 minutes under flow at each shear stress using ImageJ software. E) Flow assays: experiments at all shear stresses combined. F–H) To allow direct comparison of the results from static and flow assays, the rosetting values from each experiment were converted to a percentage of the “no additive” control value. F) IT/R29 parasites with 100 µg/ml heparin. G) IT/R29 parasites with 10 µg/ml heparin. H) IT/R29 parasites with 1 µg/ml heparin. Mean and standard error from three independent experiments (A–D and F–H) or nine experiments (E) are shown. Mean values that are statistically significantly different from the control value (A–E) or the static value (F–H) by one- way ANOVA with Tukey’s post test are shown by asterisks, *p<0.05, ***p<0.001.