Alp activity assay kit

ALP activity was recorded at 405 nm using an ALP activity assay kit (Jiancheng, Nanjing, China) as previously described^{14 (link)}. Total protein content of each sample was determined with a BCA kit (Beyotime, China). ALP activity relative to the control group was normalized to the total protein content.
According to the protocol of the NBT/BCIP staining kit (Beyotime, China, transfected SCAPs were washed with PBS and fixed in 4% PFA for 30 min. After PBS wash for three times, cells were incubated in alkaline solution for 20 min at 37 °C.

The experiment was performed after osteogenic induction for 5 days of transfected SCAPs. ALP staining kit (Beyotime, China) is used to qualitatively detect the expression of ALP as described previously [35 (link)]. ALP activity was examined with an ALP activity assay kit (Jiancheng, Nanjing, China). The absorbance was detected in the microplate reader at 520 nm. ALP activity was normalized to the total protein level.

BMSCs were induced and cultured in osteogenic medium according to a StemPro Osteogenesis Differentiation Kit (Invitrogen, USA). Recombinant human BMP-4 protein (rhBMP-4) (R&D Systems, USA), a Smad inhibitor (LDN-193189) (Selleck, USA), and metformin (TargetMol, USA) were separately added to 0.5 ml medium in 24-well dishes (Corning, USA), and the final concentrations of each substance in the medium were as follows: rhBMP-4 (+: 10 ng/ml; ++: 50 ng/ml), LDN-193189 (+: 0.1 μM; ++: 0.5 μM), and metformin (+: 30 μM; ++: 100 μM). After 10 d of induction, cells were fixed in 70% ethanol for 1 h and stained using an ALP staining kit (Beyotime, China) according to the manufacturer's protocol. Intracellular ALP activity assays of BMSCs were performed at 3, 5, and 7 d of induction using an ALP Activity Assay Kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's protocol and were standardized based on protein concentration. After induction for 21 d, cells were fixed in 70% ethanol and stained with 2% alizarin red S staining solution (Sigma-Aldrich, USA) for 5 min. Then, 1 ml of isopropanol was added into each well to dissolve the red perylenequinone derivatives in the calcium nodules, and the optical density (OD) values were measured at a wavelength of 550 nm.

On days 3, 7, 14, and 21, cells were lysed with RIPA buffer for 30 min on ice, and the protein concentration was measured according to the instructions of a bicinchoninic acid protein assay kit (CWBIO, Jiangsu, China). The activity of ALP (at days 3, 7, and 14), an early osteogenic marker (Birmingham et al., 2012 (link)), was detected following the instructions of an ALP activity assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the absorbance at a wavelength of 520 nm was measured with a microplate reader (SPECTROstar Nano, Germany). The calcium content (at day 21) in the extract was used as a late osteogenic marker (Salim et al., 2004 (link)) and measured colorimetrically using a calcium quantification kit (Nanjing Jiancheng Bioengineering Institute). Then, the absorbance at a wavelength of 620 nm was measured with a microplate reader.

The experiment was performed after osteogenic induction for 7 and 14 days of cultured BMSCs within membranes. ALP staining was processed using an ALP staining kit (Beyotime, China). Quantification of ALP activity was determined with an ALP activity assay kit (Jiancheng, China). Absorbance was read at 520 nm.

Osteogenic differentiation of MC3T3-E1 cells was studied by measuring the alkaline phosphatase (ALP) activity and ALP staining in DMEM medium (supplemented with 10% FBS and 1% P/S) and in a medium supplemented with osteoinductive factors (10⁻⁸ mol/L dexamethasone, 10 mmol/L β-glycerophosphate and 50 µg/mL ascorbic acid; all purchased from Sigma).
ALP expression was measured using the ALP activity assay kit (JianCheng Biotech, Nanjing, China) in accordance with the manufacturer’s instructions. For the analysis of ALP activity, cell lysates were isolated using a cell lysis buffer on day 7, and ALP substrate buffer containing 5 mmol/L of p-nitrophenol (pNP) was added to each group. After incubation at 37 °C for 15 min, the absorbance at 520 nm was measured with a microplate reader. The ALP activity value was normalized by protein content measured at 562 nm.
The ALP staining process was as follows. First, the samples were fixed in 4% (w/v) paraformaldehyde for 30 min, and then dye solution was added and the samples incubated in the dark for 1 h. In the final step, the dyeing conditions of the MC3T3-E1 cells on the samples were recorded by a digital camera.