Pnpp alkaline phosphatase assay kit

Cell-laden microfibers
after 7 days in culture were washed with 0.9% NaCl, soaked in 50 mM
EDTA and 0.2 mg/mL collagenase solution in 0.9% NaCl for 10 min at
room temperature and then incubated in trypsin solution for 10 min
to retrieve cells from the microfibers. The obtained cells were washed
in 0.9% NaCl, counted, and lysed for 30 min in a lysis buffer (0.1%
Triton X-100, 50 mM Tris-HCl) at 4 °C. The ALP activity was measured
with a pNPP Alkaline Phosphatase Assay Kit (Sigma) according to the
user’s manual. Bovine ALP (Sigma) was adopted to create a standard
curve. Absorbance determined at 405 nm was read by a fluorescent plate
reader (BioTek, USA) and normalized to cell counts.

Utilizing the pNPP Alkaline Phosphatase assay kit (Sigma-Aldrich, St. Louis, MI, USA), the alkaline phosphatase (ALP) activity was measured 7, 14, and 21 days after osteogenic differentiation. Briefly, the medium was withdrawn, each membrane was transferred to the new plate and the cell layers were washed three times with PBS and permeabilized overnight at 4 °C with 0.1% Triton X-100 at designated times post-induction. The following day, 50 μL of lysate was added to a 1 M diethanolamine buffer (pH 9.8, containing 0.5 mM MgCl₂) containing 1 mg/mL pNPP (4-nitrophenyl phosphate disodium salt hexahydrate). In each well, one hundred microliters of pNPP substrate were introduced. The plates were incubated for 30 min at room temperature in order to produce the yellow, water-soluble reaction product. The reaction was terminated by the addition of 3 M NaOH, and the absorbance at 405 nm was measured using an ELISA microplate reader (RT-2100c, Rayto, China). Each well’s ALP activity was adjusted to its total protein content. BioSpec-nano (Shimadzu, Kyoto, Japan) was used to measure the total protein content.