Isolation of neurospheres from primary mouse tissue and of <i>in vitro</i> cultured neurospheres using <i>Sox2-</i>MB.

<p>(A) Two cell populations, namely <i>Sox2-</i>MB^high and the <i>Sox2-</i>MB^low, were first selected on Annexin-V^- cells and then by comparing the nonspecific-MB fluorescent signal to the Sox2-MB fluorescent signal. (B) After 1 wk, sphere-forming efficiency was calculated from the <i>Sox2-</i>MB^high and the <i>Sox2-</i>MB^low populations as well as non-sorted primary mouse hippocampus isolated cells. (C and D) Images of 1 wk old spheres generated from sorted <i>Sox2-</i>MB^low cells and <i>Sox2-</i>MB^high cells (scale bar = 25 µm). (E) Neurospheres from the <i>Sox2-</i>MB^high and the <i>Sox2-</i>MB^low populations were serially passaged and cumulative population doublings was calculated. (F) <i>In vitro</i> cultured neurosphere mRNA expression of <i>Sox2</i> was analyzed by RT-PCR and compared to MEFs. (G) Two cell populations, namely <i>Sox2</i>-MB^high and <i>Sox2-</i>MB^low, were selected by comparing the nonspecific-MB fluorescent signal to the <i>Sox2-</i>MB fluorescent signal. (H) After 1 wk, sphere-forming efficiencies were calculated. (I) Neurospheres formed by the <i>Sox2-</i>MB^high and the <i>Sox2-</i>MB^low populations were stained for Sox2 and Nestin, or secondary antibodies only (control) (scale bar = 50 µm). Error bars represent the mean ± SEM. Asterisks denotes statistical significance ((n = 5 samples ^*<i>p</i><0.05, n = 3 samples^***<i>p</i><0.001).