Delta1 dose-dependent induction of Notch target genes Hes1 and Hes5 and Hes5 is required to accurately detect different levels of Notch signaling induced by different densities of Delta1^ext-IgG. (A-C) RNA was prepared from uncultured LSKSP cells or from LSKSP cells incubated for 3 hours (A), 7 days (B), and 14 days (C) in wells precoated with Delta1^ext-IgG solutions (ranging from 10 μg/mL) or human IgG1 solutions (10 μg/mL; depicted as 0). Hes1 and Hes5 mRNA levels were determined by SyBR green quantitative RT-PCR. Results are normalized to the housekeeping gene Rpl7 and are shown either as fold-increase (A) in cultured cells versus uncultured cells or as absolute values. Each bar represents the mean (± SEM) of 3- replicates. (B,C) Generated slopes are significantly different from 0.0 (P < .05). (D) After incubation of sorted LSKSP from Hes5^+/+ or Hes5^−/− littermates with different densities of Delta1^ext-IgG, sorted B220⁺ cells were transferred to OP-9 stromal layers. Wells were designated positive if more than 1% of the cells growing on OP-9 stromal layers were B220⁺CD19⁺. (E) Plot showing results of a single dose using limit dilution to calculate a frequency for B-cell progenitors. In this representative example, the calculated frequency of B-cell progenitors in Hes5^+/+ is 490 per 10⁶ B220⁺ starting cells and for Hes5^−/− is 3205 per 10⁶ B220⁺ starting cells.