Proteins were extracted from the cells, separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat dry milk and incubated overnight at 4°C with primary antibodies against the following proteins: GSK3β, p-GSK3β (Ser 9), DKK3, β-catenin, cyclin D1, Bcl-2, and Bax-1. These antibodies were purchased from Abcam (Shanghai, China). An anti-GAPDH or β-actin antibody obtained from Sigma-Aldrich was used as a loading control. The resulting bands on the immunoblots were visualized using a BCIP/NBT kit (Sigma, St. Louis, MO, USA). The band intensities from the western blotting experiments were quantified with image analysis software (ImageQuant TL; Amersham Biosciences) prior to the statistical analysis.
Bcip nbt kit
Manufactured by Merck Group
Sourced in United States
The BCIP/NBT kit is a laboratory reagent used for the detection and visualization of alkaline phosphatase (AP) activity in various biological applications, such as immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assays (ELISA). The kit contains the substrates BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitro blue tetrazolium), which, when combined with AP, produce a colorimetric reaction that results in a dark purple or blue precipitate at the site of AP activity.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant tl, by Cytiva (2 mentions)
Thiazolyl blue tetrazolium blue mtt assay, by Merck Group (1 mentions)
Sigmafast p nitrophenyl phosphate kit, by Merck Group (1 mentions)
Stempro differentiation media, by Thermo Fisher Scientific (1 mentions)
Oil red o kit, by Merck Group (1 mentions)
Alcian blue staining solution, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
6 protocols using bcip nbt kit
1
Western Blot Analysis of Cell Signaling
2
Quantifying Alkaline Phosphatase Activity
3
Alkaline Phosphatase-Based Osteogenesis Assay
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In vitro calcification was determined by alkaline-phosphatase (ALP) activity, which is an early marker of osteogenic differentiation. ALP staining was performed using Sigma BCIP®/NBT kit 14 days after the initiation of differentiation. Cells were washed with PBS and incubated with alkaline-phosphatase working solution for 10-15 min at room temperature. ALP activity appeared as blue deposition and plates were photographed with digital camera.
4
Trilineage Differentiation of hUC-MSCs
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For all differentiation assays, hUC-MSCs were first cultured in microcryogels for 0, 3, and 5 d before being digested, and were subsequently seeded in six-well plates. Cells were cultured in growth medium for 12 h followed by changing to differentiation medium. The trilineage differentiation ability of hUC-MSCs was assessed by culturing the cells in StemPro® differentiation media (Gibco, Life Technologies, USA) to induce osteogenesis, chondrogenesis, and adipogenesis. Osteogenesis was assessed at 14 d by staining the cells for alkaline phosphatase with the BCIP/NBT Kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Chondrogenesis was assessed at 14 d by staining for sulfated proteoglycan deposits with Alcian-Blue Staining Solution (Sigma-Aldrich). Adipogenesis was assessed at 21 d by staining for lipid accumulation with an Oil Red O Kit (Sigma-Aldrich).
5
Protein Expression Analysis by Western Blot
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Proteins were separated through using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes. We used 5% skim milk powder to block the membranes. The membranes were incubated overnight at 4 °C with primary antibodies against the following proteins: PLXNB2 (Cat. ab229950; Abcam, Shanghai, China), cyclin D1(Cat. ab16663; Abcam, Shanghai, China), B-cell lymphoma-2 (BCL2, Cat. ab182858; Abcam, Shanghai, China), and BCL2-associated X protein (BAX, Cat. ab32503; Abcam, Shanghai, China). We used the anti-β-actin antibody (Cat. ZRB1312; Sigma, St. Louis, MO, USA) as the loading control. Bands on the Western blots were visualized using the BCIP/NBT kit (Sigma-Aldrich, St. Louis, MO, USA). Image analysis software (ImageQuant TL; Amersham Biosciences, USA) was used to quantify the band intensities on the immunoblots.
6
Alkaline Phosphatase Staining Protocol
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Cells were fixed and washed using the same protocol as the AR staining described in the previous section and the fixed cells were incubated with a staining mixture for ALP provided in the BCIP/NBT kit (Sigma-Aldrich, St. Louis, MO, USA) for 45 min at 37 °C in dark conditions following the kit protocol. Once the color developed, the dye was removed and stained monolayers were washed 2 times and covered with milli-Q water (1 mL/well) and photographed using a Lionheart XF Automated Microscope (Biotek, Winooski, VT, USA).
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