Analysis of differentiated hESCs by image scanning flow cytometry. hESCs allowed to differentiate on S17 stromal cells were analyzed by image scanning flow cytometry to provide concurrent data on cell phenotype and morphology. Cells were stained for (A) CD34 and CD31 and (B) CD34 and CD45 with cell size distribution of cells gated for CD34^brightCD31⁺ and CD34^dimCD45⁺ cells as indicated. Mean signal intensity for each cell population is shown. (C,D) Images of cells gated for (C) CD34^bright and (D) CD34^dim expression. Brightfield (BF) channel indicates cell morphology, side scatter (SSC) shown in blue indicates granularity, and exclusion of 7-AAD is illustrated by absence of red staining. Pink staining is indicative of CD34 expression, and orange color demonstrates expression of indicated cell-surface antigens.