Gene expression analysis in CD31⁺/CD34⁺/c-Kit⁺ AGM cells separated by CD45 expression.

<p>(<b>A</b>) Single cell suspensions of the caudal portion of embryos containing the AGM region at 10.5 dpc were prepared and analyzed by flow cytometry. Cells expressing CD31 and CD34, IAC markers, were first gated. The profile shows expression of c-Kit (x-axis) and CD45 (y-axis) in CD31⁺/CD34⁺ AGM cells (left). Based on intensity of CD45 expression, CD31⁺/CD34⁺/c-Kit⁺ AGM cells were separated into three fractions, CD45-negative (under 10² of CD45-fluorescence, same as negative control), -low positive (from 10².⁵ to 10³.⁵ of CD45-fluorescence), and -high positive (approximately over 10⁴ of CD45-fluorescence). Isotype control and compensation samples of flow cytometric analysis are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035763#pone.0035763.s004" target="_blank">Figure S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035763#pone.0035763.s001" target="_blank">S5</a>. (<b>B</b>) The percentage of CD45-negative, -low positive, and -high positive c-Kit⁺/CD31⁺/CD34⁺ AGM cells was calculated both at 9.5 dpc (white bars) and 10.5 dpc (black bars). (<b>C-H</b>) Gene expression of <i>CD45</i> (C), <i>Runx1</i> (D), <i>c-Myb</i> (E), <i>Evi-1</i> (F), <i>SCL</i> (G) and <i>Gata2</i> (H) was analyzed in sorted CD45-negative, -low positive and -high positive c-Kit⁺/CD31⁺/CD34⁺ AGM cells. Expression levels of <i>CD45</i> mRNA are up-regulated as c-Kit⁺/CD31⁺/CD34⁺ cells express CD45 surface protein. Expression levels of <i>Runx1</i>, <i>c-Myb</i>, <i>Evi-1</i>, <i>SCL</i> and <i>Gata2</i> were highest in CD45-low positive c-Kit⁺/CD31⁺/CD34⁺ cells, whereas that of <i>Evi-1</i> was highest in CD45-negative c-Kit⁺/CD31⁺/CD34⁺ cells. RQ represents relative quantity of template in the original sample.