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. 2000 Jun 12;149(6):1297–1308. doi: 10.1083/jcb.149.6.1297

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© 2000 The Rockefeller University Press

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graphic file with name JCB0001087.f3a.jpg — Effect of FGF on osteoblast differentiation. (A) Inhibition of alkaline phosphatase (ALP) and mineralization. Primary calvarial osteoblasts were maintained in differentiation medium for three weeks. FGF1 (10 ng/ml) and heparin were added from the start of the experiments and the differentiation medium was replaced every 3 d. Duplicate plates were fixed and stained for alkaline phosphatase enzymatic activity using Fast blue RR (Sigma) and for mineralization by Von Kossa staining. Positive cells stain purple for ALP expression and black areas indicate areas of mineralization. (B) Effect of FGF on osteoblast differentiation. 10,000 cells/well of primary osteoblasts were plated on coverslips in 10% FCS and differentiation medium was added to the wells at time 0. FGF1 was added at 10 ng/ml. BrdU (4 μg/ml) was added during the last 6 h of each time point. Cells were stained for alkaline phosphatase expression and for BrdU incorporation. Percentage of cells incorporating BrdU are shown. The percentage of cells that were positive for alkaline phosphatase were counted for each time point and are indicated on top of the graph. Data represent the mean of duplicate experiments. Bar, 800 μm.

graphic file with name JCB0001087.f3b.jpg — Effect of FGF on osteoblast differentiation. (A) Inhibition of alkaline phosphatase (ALP) and mineralization. Primary calvarial osteoblasts were maintained in differentiation medium for three weeks. FGF1 (10 ng/ml) and heparin were added from the start of the experiments and the differentiation medium was replaced every 3 d. Duplicate plates were fixed and stained for alkaline phosphatase enzymatic activity using Fast blue RR (Sigma) and for mineralization by Von Kossa staining. Positive cells stain purple for ALP expression and black areas indicate areas of mineralization. (B) Effect of FGF on osteoblast differentiation. 10,000 cells/well of primary osteoblasts were plated on coverslips in 10% FCS and differentiation medium was added to the wells at time 0. FGF1 was added at 10 ng/ml. BrdU (4 μg/ml) was added during the last 6 h of each time point. Cells were stained for alkaline phosphatase expression and for BrdU incorporation. Percentage of cells incorporating BrdU are shown. The percentage of cells that were positive for alkaline phosphatase were counted for each time point and are indicated on top of the graph. Data represent the mean of duplicate experiments. Bar, 800 μm.