A CR3⁺population of HPCs tethered to stroma-iC3b and can be observed to proliferate in response to β-glucan in a CR3-dependent manner. (A) Sorted Sca1⁺, c-Kit⁺, lin^- HPCs were stained with anti-CD11b mAb and analyzed by flow cytometry. Filled curve represents isotype control for anti-CD11b; open curve shows HPCs gated for anti-CD11b staining. (B) A short-term cobblestone assay using WT (▪) or CR3^-/- HPCs (□) was analyzed after 12 days of culture in the presence of stroma-iC3b in response to β-glucan (10 μg/mL) compared with their counterparts incubated with stroma-iC3b only. The CFU was also compared between WT and CR3^-/- HPCs in the absence of serum (no serum) and β-glucan. (C-D) Representative micrographs captured from WT HPCs grown in the presence of serum and serum and β-glucan, respectively. Arrows indicate colonies. (E) Long-term culture-initiating culture (LTC-IC), established 35 days after coculture of WT (▪) or CR3^-/- HPCs (□) and stroma in the presence of stroma-iC3b and β-glucan and analyzed 35 days later in methylcellulose culture compared with their counterparts incubated with stroma-iC3b only. Data in panels B and E represent the mean ± SEM number of CFUs observed in triplicate wells in experiments that were repeated twice (^*P < .05 and ^**P < .005). HPCs were sorted from 3 WT or CR3^-/- mice and then plated with stromal cells. The data shown here are 1 representative experiment of 3 separate experiments (total n = 9 in each group).