Specific expression of ESAM on HSC-enriched fraction of E14.5 fetal liver. Flow cytometric analysis was performed for Rag1/GFP⁻ cells of E14.5 fetal liver using anti-c-kit, anti-Sca1, and anti-ESAM Abs. First, Rag1/GFP⁻ cells were sorted from E14.5 fetal liver of Rag1/GFP knockin heterozygous fetuses with high purity. The sorted cells were incubated with a purified rat anti–mouse ESAM Ab (1G8) followed by goat anti–rat IgG-FITC. The cells were then stained with anti-c-kit-APC, anti-Sca1-PE, and 7-AAD. To minimize the nonspecific binding of anti-c-kit and Sca1 mAbs to the cells wearing goat anti–rat IgG-FITC, the cells were incubated with a rat anti–mouse FcRII/III Ab before the anti-c-kit and Sca1 staining. (A) The Rag1/GFP⁻ cells were analyzed with respect to expression of ESAM, c-kit, and Sca1 (Left, middle). Expression of c-kit in the Sca1^Hi ESAM^Hi cells (middle, inset) is presented (right). (B) The conventional c-kit^Hi Sca1⁺ fraction (left, inset) could be divided into 2 fractions, ESAM^−/Lo and ESAM^Hi (middle). The cells were stained with an isotype control IgG (dashed line) or with the anti-ESAM Ab (solid line). The ESAM^Hi cells (yellow) were found as c-kit^Hi Sca1^Hi, whereas the ESAM^−/Lo cells (pink) were c-kit^Hi Sca1^Lo (right). (C) Six-color flow cytometric analysis using an anti-ESAM Ab followed by goat anti–rat IgG-FITC, a PE-anti-CD48 Ab, biotin-anti-lineage marker Abs (TER119, Gr1, CD3, CD45R/B220) followed by SA-PETR, a PE-Cy7-anti-Sca1 Ab, an APC-anti-c-kit, and 7-AAD was performed for E14.5 fetal liver cells of WT C57B6 embryos. The profile of Lin⁻ cells regarding c-kit and Sca1 expression is shown in the left. The Lin⁻ c-kit^Hi Sca1^Lo and Lin⁻ c-kit^Hi Sca1^Hi fractions gated in the left panel were analyzed with respect to expression of ESAM and CD48 (middle and right). The percentage of cells in each gate is indicated in each panel.