Disodium p nitrophenyl phosphate

Cells were plated in 96-well plates at a density of 1 × 10‍⁴ cells/well, then cultured for 72 h in the above-mentioned medium containing BMP-2 (100 ng/ml), in the presence and absence of either 8-nitro-cGMP or 8-bromo-cGMP (30 μmol/L). Cells were fixed for 30 min in 4% paraformaldehyde and washed with PBS, then incubated for 30 min at 37°C with 100 mmol/L Tris-HCl buffer (pH 8.5) containing 270 μmol/L naphthol AS-MX phosphate (Sigma-Aldrich) and 1.4 mmol/L Fast blue BB (Sigma-Aldrich). After washing with tap water, they were observed under a microscope.
Cells were cultured in the same conditions described above, washed with PBS, and homogenized with 1% Nonidet P-40 (50 ‍μl) under sonication on ice. Cell lysates (10 μl) were added to 50 μl of 0.2 mol/L Tris–HCl buffer (pH 9.5) containing one mmol/L MgCl₂ and 12.5 mmol/L disodium p-nitrophenyl phosphate (Fujifilm Wako Pure Chemical Co.). After incubation for 15 min at 37°C, the reactions were terminated by adding 50 μl of 0.5 mol/L NaOH, and the absorbance of the reaction mixture at 405 nm was read using a microplate reader (SH-1000; Corona Electric, Ibaraki, Japan). The increase in absorbance after 15 min was divided by the amount of cellular protein, with the obtained value used to express the specific activity of ALP.