CD45⁺-cell depletion from primary OBL cultures blocks the inhibitory effects of IL-3. (A) Murine BM was cultured in α-MEM plus 10% FCS media for 8 days as described in “Materials and methods.” These cells were the starting population for further OBL differentiation studies. Flow cytometry analysis shows a PE-CD45^- population of approximately 70% and a PE-CD45⁺ population of 30%. CD45⁺ cells were then depleted from the total population by magnetic bead depletion as described in “Materials and methods.” The resulting population was essentially 100% CD45^- by flow cytometry. The CD45⁺-enriched population was recovered after depletion and was approximately 25% CD45^- and 75% CD45⁺. (B) The CD45-depleted or reconstituted cell populations or control cells were plated as usual in osteogenic media with varying concentrations of IL-3 or TNF-α (0.1 to 10 ng/mL), and ALP activity was determined as usual after 10 days in culture. ▪ represents cells depleted with a nonspecific IgG primary antibody; ▧, CD45-depleted cells; and ▤, CD45-depleted cultures reconstituted with CD45⁺ cells. Depleting CD45⁺ cells results in a cleaner stromal-cell/OBL population, so the ALP level per well was approximately 2 times control antibody depletion. Data are represented as percent of media alone (without IL-3 treatment) for each group and are mean ± standard deviation for 3 wells for each concentration for a typical experiment. Similar results were seen in 2 independent experiments. ^*P < .05; N.S. indicates not significant. (C) CD45⁺ cells were depleted from human BM cell cultures by immunomagnetic method using coated anti-CD45 microbeads and treated with IL-3 (20 ng/mL) for the entire culture period. Alkaline phosphatase staining was performed after 14 days and quantified by 1D Image Analysis Software (Kodak Digital Science). CD45⁺ cell depletion resulted in loss of the IL-3 inhibitory effects in CFU-F cultures. Data represent the average ± standard deviation of 3 wells for each condition for a typical experiment.