Alkaline phosphatase yellow pnpp liquid substrate system for elisa

Alkaline phosphatase (ALP) staining was performed after 5 days of differentiation of primary osteoblasts using the BCIP/NBT Liquid Substrate System (B1911, Sigma-Aldrich). ALP activity was also measured using the Alkaline Phosphatase Yellow (pNPP) Liquid Substrate System for ELISA (P7998, Sigma-Aldrich) according to the manufacturer’s instructions. For alizarin red Staining, cells were fixed after 2–3 weeks of differentiation in 10% neutral buffered formalin and stained with a 1.36% alizarin red S (A5533, Sigma-Aldrich) solution (pH 4.1–4.3) for 1 h in the dark. For quantification, alizarin red S was extracted by incubation in a 10% cetylpyridinium chloride (CPC; C0732, Sigma-Aldrich) solution for 1 h. The collected CPC was transferred to a 96-well plate (0.2 ml/well), and the absorbance was read at 570 nm using an ELISA reader. For TRAP staining, cells were fixed in a 4% paraformaldehyde solution in phosphate-buffered saline and stained for TRAP using a leukocyte acid phosphatase kit (#387, Sigma-Aldrich) according to the manufacturer’s protocol. TRAP-positive cells with three or more nuclei were counted.

The assay was performed on a 96-well plate coated with 1 µg of GFPE-N or GFPE-C antigen per well (20 ng/μL) and 100 mM carbonate buffer pH 9.6 as background control. Immobilization was carried out at 4 °C overnight. Subsequently, each well was blocked with 250 µL of 3% BSA (Sigma-Aldrich, Saint Louis, MO, USA) in PBS for 1 h at 37 °C. Thereafter, three washes with 0.05% Tween-20 in PBS (wash buffer) were performed. TEV or TEVK VLPs were mixed with anti-TEV antibody (Agdia, Elkhart, IN, USA) in 1:100 dilution using PBS/1% BSA as a diluent, and each mixture was incubated at 37 °C/200 rpm for 2.5 h. The antibody/VLP mixture was added to the wells in triplicate, calculating 1 µg of each VLP per well. As control, anti-TEV antibody alone was added in 1:100 dilution to antigen-coated wells (data not shown). To allow interaction, the plate was incubated at 37 °C for 1 h. Subsequently, the plate was washed three times with wash buffer and once with PBS. Finally, 50 µL of Alkaline Phosphatase Yellow (pNPP) Liquid Substrate System for ELISA (Sigma-Aldrich, Saint Louis, MO, USA) was added to each well, and absorbance was read at 405 nm.

ALP activity of MC3T3-E1 cells were examined on days 4, 7 and 11. For ALP activity assay, MC3T3-E1 cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, USA) for 30 min, and then rinsed with deionized water three times. Fixed cells were treated with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP solution, Sigma-Aldrich, USA) for 30 min. ALP positive cells were visualized as deep blue under the optical microscope. For quantification of ALP activity, cells were washed once with PBS solution and 70μl Alkaline Phosphatase Yellow (pNPP) Liquid Substrate System for ELISA (Sigma-Aldrich, USA) was added. The cells were then incubated for 5 min in the dark. Finally, ALP activity was analyzed by an ELISA reader at 405nm wavelength.
Calcium deposits produced by tested MC3T3-E1 cells were examined on days 7, 14 and 21 by ARS staining assay. For Alizarin Red S staining, MC3T3-E1 cells were washed with PBS solution and fixed in 4% paraformaldehyde for 30 min. Cells were then stained with 1% Alizarin Red S (ARS, Sigma-Aldrich, USA) pH 4.1 for 30 min followed by washing with excess deionized water. For the quantification of ARS staining, stained cells were destained for 15 min with 10% cetylpyridinium chloride (Sigma-Aldrich, USA) and measured at 562 nm wavelength using a microplate reader.