Modified great escape seap chemiluminescence assay

The ALP activities were assessed quantitatively and qualitatively using the modified Great Escape SEAP chemiluminescence assay (BD Clontech) and/or histochemical staining as described previously [64 (link), 106 (link)]. Briefly, for the histochemical staining, the cells were fixed with 0.05% glutaradehyde at room temperature for 10 min. After being washed with PBS, cells were stained subjected to histochemical staining with a mixture of 0.1 mg/mL of napthol AS-MX phosphate and 0.6 mg/mL of Fast Blue BB salt. After 20 minutes, the mixture was removed and replaced with PBS. Histochemical staining was recorded using bright light microscopy. For the chemiluminescence assay, the cells were lysed by the Cell Culture Lysis Buffer (Promega, Madison, WI). Then 5μl Cell Lysis Buffer, 5ul substrate (BD Clontech) and 15μl Lupo Buffer were mixed well under a light-proof condition, and incubated at room temperature for 20 minutes before measuring chemiluminescence signals. Each assay condition was performed in triplicate. The results were repeated in at least three independent batches of experiments. ALP activities were normalized by total cellular protein concentrations among the samples.

The alkaline phosphatase (ALP) activities were examined using the modified Great Escape SEAP chemiluminescence assay (BD Clontech) and/or histochemical staining, as described before [16 (link),37 (link)]. For ALP histochemical staining, iMEF cells were induced for osteogenic differentiation using AdBMP9, and AdGFP was used as control. At indicated time points, cells were first fixed with 0.05% glutaraldehyde at room temperature for 10 min, then washed with distilled water, and finally subjected to histochemical staining with a mixture containing 0.1 mg/ml of napthol AS-MX phosphate and 0.6 mg/ml of Fast Blue BB salt and stored in the dark for 10 min. Histochemical staining was recorded with the use of bright light microscopy.
For the chemiluminescence assays, iMEFs were first lysed by cell lysis reagent (Promega, U.S.A.), and then 5 μl of the cell lysis liquid, 5 μl substrate (BD Clontech), and 15 μl Lupo buffer were fully mixed and stored in the dark at room temperature for 20 min. ALP activities were normalized by total cellular protein concentrations in each sample. Each assay condition was performed in triplicate, and the results were repeated in three independent experiments.