Figure 6

Enhanced osteoblast development by salubrinal in the CFU-OBL assay. Bone marrow-derived cells were plated at 2 × 10⁶/ml in 6-well plates in osteogenic differentiation medium supplemented with 10 nM dexamethasone, 50 μg/ml ascorbic acid 2-phosphate, and 10 mM β-glycerophosphate. Cells were cultured for 2 weeks in the presence and absence of 0.5 μM salubrinal, and medium was changed every other day. For alkaline phosphatase (ALP) staining, cells were fixed in citrate-buffered acetone for 30 s, incubated in the alkaline-dye mix for 30 min, and counterstained with Mayer’s Hematoxylin for 10 min. Cells were then evaluated microscopically and the intensity of ALP staining was determined. The images display 2 pairs of osteoblast cultures.