Alp assay kit

Rat tail type I collagen, Transwell inserts (0.9 cm² cell culture area, 1.6 × 10⁶ pores/cm²), 12-well polystyrene tissue culture dishes, 12-well polycarbonate plates and Transwell inserts (1.12 cm² cell culture area, 1.6 × 10⁸ pores/cm²) were purchased from Corning (Corning, NY). SB202190 was from Selleckchem (Houston, TX). Collagenase (type IV) was purchased from Worthington Biochemicals (Lakewood, NJ). Y-27632 and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) were purchased from ApexBio Technology (Houston, TX). Human collagen (type I) was obtained from Advanced Biomatrix (Carlsbad, CA). The ALP assay kit was from AbCam (Cambridge, MA). Gastrin was from Anaspec (Freemont, CA). UGT-Glo™ Assay and P450-Glo™ CYP3A4 Assay were obtained from Promega (Madison, WI). N-acetyl cysteine was purchased from MP Biomedicals (Santa Ana, CA). Murine EGF was procured from Peprotech (Rock Hill, NJ). Primocin was purchased from InvivoGen (San Diego, CA). Nicotinamide, L-alanine p-nitroanilide, fluorescein diacetate, loperamide, zafirlukast, cobalt chloride, potassium phosphate buffer, bovine serum albumin, and A83–01 were acquired from Sigma-Aldrich (St. Louis, MO). All other reagents and Bicinchoninic Acid (BCA) Protein Assay Kit were purchased from Thermo Fisher Scientific (Waltham, MA).

ALP activity was determined following the manual instructions of ALP assay kit (Abcam plc, Cambridge, UK). The color of the reaction was measured at 405 nm. The number of viable cells was determined using the Cell Titer-Blue cell viability assay according to the manufacturer’s instructions (Promega, USA) at OD 579. ALP activity was represented as fold change over control non-induced cells after normalization to the number of viable cells [40 (link)].

Human endometrium cells (Ishiwaka) were cultured in MEM without phenol red with Earle’s Salts culture, which was supplemented with estrogen-deprived 10 DCC-FBS du-ring 5 days [38 (link)]. Then, 100000 cells were plated per well in 12-well plates to be treated with VEH (0.05% DMSO), test compounds (0.3–10 μM), ICI (0.1 μM) and 4-OHTAM (0.3–3 μM), for 24, 48 and 72 h, in the absence or in the presence of E2 (10 nM). Cells were washed with ice-cold PBS, and Alkaline Phosphatase (ALP) activity was measured by using ALP Assay Kit (Abcam, Cambridge, MA, USA). The substrate p-nitrophenyl phosphate (pNPP) and the ALP enzyme were used to detect ALP activity by the analysis of the optical density at 405 nm in the iMark microplate Reader (Bio-Rad, Irvine, CA, USA). Maximal ALP activity from pure agonist E2 was expressed as Emax (100%), which allowed the determination of EC₅₀ and IC₅₀ values. Protein concentration was quantified with the bicinchoninic acid assay (BCA) kit (Bio-Rad).

hMSCs were collected and washed. The cells were lysed by 1% Triton X-100 for 15 min and centrifuged at 10,000 g for 5 min. The supernatant was used for ALP analysis by ALP Assay Kit (Abcam, USA).

MC3T3-E1 cells were incubated in 24-well plates at 5 × 10³ cells/well and allowed to adhere overnight. The medium was replaced, and the cells were treated with osteogenic differentiation medium and different concentrations of HP for 14 days. All cell lines were stained with alkaline phosphatase (ALP) assay kit (Abcam, USA).

Experimental cells were harvested at different time points and washed with cold PBS three times. Cells were suspended in 500 µL of assay buffer and homogenized using a homogenizer. Cell lysates were centrifuged at 12000 g, 4 °C for 15 min. The supernatants were collected and stored on ice for further assay. The alkaline phosphatase activity of samples was measured using the ALP assay kit (Abcam, Cambridge, MA, USA).