Fast bcip nbt

Cells were seeded at a density of 4.5 × 10⁴ cells/well in 6-well cell culture plates. At 70 – 90% cell confluence, cells were treated with 50 μM quercetin for 96 h, or were transfected with mirVana™ mimics of hsa-let-7c or a non-coding miR control (miR-NC) for 96 h (Thermo Fischer Scientific, Waltham, MA USA) using lipofectamine RNAiMax reagent (Life Technologies, Darmstadt, Germany), or were left untransfected. The cells were washed, fixed with methanol, washed and incubated with FAST BCIP/NBT (Sigma-Aldrich, St. Louis, Missouri, USA), washed, and the deep bluish purple color of alkaline phosphatase expressing osteoblasts was visualized under 10× magnification using an inverted microscope (Nikon Eclipse TS100; Nikon GmbH, Düsseldorf, Germany). The Sigma FAST BCIP/NBT active substrate solution containing BCIP (0.15 mg/ml), NBT (0.30 mg/ml), Tris buffer (100 mM), and MgCl2 (5 mM), pH 9.25–9.75 was prepared by dissolving one tablet in 10ml of water.

After 7, 16 and 21 days of osteoinduction, 2 mL of the culture medium were collected for the alkaline phosphatase and osteocalcin assays. A colorimetric assay was used for the determination of ALP activity (calcium kit, Sigma and Procedure n° 0150, Stanbio Laboratory) and osteocalcin, the Osteocalcin ELISA kit (Takara BioInc., Shiga, Japan) according to the instructions of the manufacturer [14 (link)]. ALP staining on hASC and osteoblasts (16 day of osteoinduction), was done with FAST BCIP/NBT (Sigma-Aldrich, St. Louis, Missouri, USA) (Catalog number B5655), after fixing the cells with methanol according to the manufacturer´s instructions. Sigma FAST BCIP/NBT active substrate solution containing BCIP (0.15 mg/ml), NBT (0.30 mg/ml), Tris buffer (100 mM), and MgCl2 (5 mM), pH 9.25–9.75 was prepared by dissolving one tablet in 10ml of water. The BCIP-NBT substrate is hydrolyzed/reduced by ALP, when osteoblast positive, to form a deep-purple/black precipitate which is readily detectable by eye and was photographed in Olympus microscope (U-TV0.63XC).

For osteogenic differentiation, cells were seeded at a density of 4.5 × 10⁴ cells/well in 6-well cell culture plates in ready-to-use NH OsteoDiff medium (Miltenyi Biotec, Bergisch Gladbach, Germany). Ten days later, alkaline phosphatase expressed by osteoblasts was visualized using FAST BCIP/NBT (Sigma), which produces a deep blue color change.