The influence of CD45⁺ cells on the yield of CFU-F and CFU-O development from CD45^– cells. (A) Representative flow cytometric histogram plots demonstrate the distribution of both CD45^– and CD45⁺ cell populations from input BM-derived cells. (i) Day 0 BM-derived cells were incubated with saturating concentrations of CD45-PE and (ii) the CD45^– cells were recovered. R1 represents the isotype control gate corresponding to the CD45^– cell fraction, and R2 represents the region used to sort CD45⁺ cells. The purity of the recovered CD45^– fraction was more than 99%, as determined by flow cytometry. (B) CFU-F and CFU-O development from unsorted and CD45^– suspension-derived cells. Cells harvested from day 7 suspension cultures initiated with unsorted and CD45^– cells were plated in CFU-F and CFU-O assays. CFU-F assays were stained with Giemsa to visualize discrete colonies of fibroblastic cells (i,iii), and CFU-O assays (ii,iv) were incubated with tetracycline to visualize newly formed bone nodules (seen as white areas) under UV-fluorescence microscopy. Scanning electron micrographs revealed a layer of globular accretions (v) and mineralized extracellular matrix (vi). Field width (FW) for panels v and vi = 70 μm and 14μm, respectively. Note: CFU-F and CFU-O assays were initiated with 1 × 103 cells/cm2. (C) The calculated fold expansion in total cells, CFU-Fs, and CFU-Os attained in SSCs initiated with unsorted, CD45^–, and CD45⁺ cells. Calculated (i) total cell, (ii) CFU-F, and (iii) CFU-O expansions as a function of suspension culture time. The cells harvested from the CD45⁺ suspension cultures did not give rise to CFU-Fs and CFU-Os. Statistically significant differences (P < .05) are denoted with an asterisk. Each bar represents the mean ± SD (n = 3, performed in triplicate).