A: Forward scatter (FSC) / side scatter (SSC) plots of hESC, P0 and P1 RPE. hESC and RPE lie in slightly different positions on this plot, with RPE being smaller (FSC) and more granular (SSC) than hESC.B: Overlays of IgM (black) and TRA-1-60 (red) staining for hESC, P0 RPE and P1 RPE. Positive staining is clearly visible for hESC, whereas the isotype control (IgM, negative) and TRA-1-60 samples clearly overlay for both P0 and P1 RPE. Percentages indicate the percentage of positive events occurring within the gate shown (M12, M18 or M16 in hESC, P0 and P1 RPE plots respectively).C: Percentage positive events recorded for IgM isotype control and TRA-1-60 in hESC, P0 RPE and P1 RPE. Average values are hESC 0.08% IgM, 93.5% TRA-1-60; P0 RPE 0.10% IgM, 0.30% TRA-1-60 and P1 RPE 0.09% IgM and 0.32% TRA-1-60. All values except for the TRA-1-60 stained hESC are below the lower limit of detection (1.03%).D: An example of the 0.3% of TRA-1-60 events that show slightly higher staining than the isotype control in P1 RPE (even though these are within the background limits of the assay) displayed on a FSC/SSC plot. (For example taking all TRA-1-60 events in gate M16 in figure 2B and displaying them as FSC/SSC). This is compared to a representative hESC FSC/SSC plot to show that the majority of these events are separated from hESC based on SSC ie they are more granular and very unlikely to be hESC cells.