Fig 9.

High versus low surface exposure of EhMSP-1 is not clonal and can be synchronized by serum starvation. (A and B) Clones were selected from cell culture in agar, and surface EhMSP-1 exposure was measured by flow cytometry. (A) Plate showing E. histolytica clones grown in agar suspension. (B) Surface EhMSP-1 staining for selected clones. Clones isolated by growth in agar were recovered in broth culture, and EhMSP-1 on the surface of nonpermeabilized cells was stained and analyzed by flow cytometry. Representative fluorescence-activated cell sorting histograms for four clones and background staining (preimmune serum; dashed line) are shown. The percentage of cells with high surface EhMSP-1 levels and the gate used is shown on each histogram. A mixed population of cells with high and low EhMSP-1 surface exposure was recovered in each case. (C) Serum starvation results in synchronized EhMSP-1 surface exposure. E. histolytica trophozoites were serum starved for 21 h, followed by readdition of serum. Representative fluorescence-activated cell sorting histograms are shown, displaying relative EhMSP-1 surface staining immediately following (0 h) and 10 h after readdition of serum. The dashed line shows staining with preimmune serum. Following serum starvation, a single peak which persisted for more than 10 h was observed at every time point. (D) EhMSP-1 surface exposure varies with time. EhMSP-1 surface exposure was synchronized by serum starvation followed by refeeding as for panel C. EhMSP-1 surface exposure was measured by immunostaining and flow cytometry every 2.5 h. The graph shows mean fluorescence intensity versus time.