Figure 4

Flow cytometry histograms of lectin binding in unstained and stained HESCs. A-D shows histograms of SSEA-4 binding and representative lectins that were used in this study. To validate that double staining can be performed without signal interference, we determined that SSEA-4 expression was not found in the FL1 channel. Figure 4A shows a histogram plot with the overlay image of SSEA4 (black tracing) matching the histogram plot of unstained cells (grey fill). Figure 4B shows a positive peak shift in the histogram overlay with Tomato lectin (TL) in black tracing and unstained cells (grey fill). Figure 4C shows lack of Lotus tetragonolobus lectin (LTL) (black tracing) binding in the histogram overlay with unstained cells (grey fill). Figure 4D shows a histogram overlay with MAA (black tracing) binding and shows two peaks -one representing a large population that overlays unstained cells and a smaller population denoted by arrow that shows a smaller population of MAA+/SSEA4+ cells. Figure 4E shows plots of unstained hESCs, and Figure 4F shows subpopulations of cells characterized as SSEA-4+/MAA- or as SSEA-4+/MAA+ cells (arrow).