The cells were seeded in 96-well plates with 2x103 cells/well according to the following groups: control, induce, only BMP-2, BMP-2+EGF, BMP-2+FGF, BMP-2+PDGF, and BMP-2+VEGF. The treatment concentrations of BMP-2 and the other factors were 250 ng/ml and 10 ng/ml, respectively, and the same condition was applied for the rest of experiments. The cells were cultured further in the differentiation media with or without appropriate factors for 3, 7, or 14 days. After washing with PBS (Gibco, USA), the cells were lysed with 100 µl of 0.02% Triton X-100 (Sigma, USA) solution. ALP activity was monitored by colour change of p-NPP to p-nitrophenol, measured at 405 nm. The enzyme activity was normalized by total protein concentration determined through the Bradford assay and calculated as nM/min/mg of protein. For ALP staining, the cells were seeded in 24-well plates with 2x104 cells/well according to the groups and treated for 3, 7, or 14 days. After washing with PBS, the cells were fixed with 10% formalin for 30 seconds and incubated with 0.25% naphthol AS-MX phosphate alkaline (Sigma, Germany) including fast blue RR salt (Sigma-Aldrich, Brondby, Denmark) for 30 minutes.
Naphthol as mx phosphate alkaline
Manufactured by Merck Group
Sourced in Germany, United States
Naphthol AS-MX phosphate alkaline is a laboratory chemical used as a substrate in various biochemical and cellular assays. It is a chromogenic substrate that undergoes a colorimetric reaction when cleaved by alkaline phosphatase enzymes, producing a colored product. This chemical is commonly used in histochemical staining and enzyme-linked immunosorbent assays (ELISAs) to detect and quantify the presence of specific target analytes.
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Lab products found in correlation
Fast blue rr salt, by Merck Group (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Mayer s hematoxylin, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
β glycerol phosphoric acid, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Eclipse ts100, by Nikon (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Fast violet solution, by Merck Group (1 mentions)
3 protocols using naphthol as mx phosphate alkaline
1
BMP-2 Osteogenic Differentiation in Vitro
2
Osteogenic Differentiation of MB-MSCs
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MB-MSCs were seeded at 4 × 105 cells/well in a 6-well chamber slide with 3 mL media per well. After cells adhered overnight, the medium was replaced with osteogenic medium containing DMEM-F12, 10% FBS, 10−8 mol/L dexamethasone, 10 mmol/L β-glycerol phosphoric acid, and 100 mmol/L ascorbic acid (Sigma-Aldrich St. Louis, MO, USA), and cultures were continued for another 7 days.
The osteogenic capacity of MB-MSCs was evaluated by ALP staining. Cells were fixed in 4% paraformaldehyde for 30 s at room temperature, washed with water three times, and then incubated for 30 min with staining solution, which was prepared by dissolving Fast Blue RR Salt (Sigma-Aldrich, St. Louis, MO, USA) in 48 mL of ddH2O and 2 mL of Naphthol AS-MX Phosphate Alkaline (Sigma-Aldrich, St. Louis, MO, USA). Then, cells were washed with PBS and incubated for 30 min with Mayer's Hematoxylin (Sigma-Aldrich, St. Louis, MO, USA). The stained cells were washed and imaged under microscope. Purple staining indicated the synthesis of ALP by osteoblasts.
The osteogenic capacity of MB-MSCs was evaluated by ALP staining. Cells were fixed in 4% paraformaldehyde for 30 s at room temperature, washed with water three times, and then incubated for 30 min with staining solution, which was prepared by dissolving Fast Blue RR Salt (Sigma-Aldrich, St. Louis, MO, USA) in 48 mL of ddH2O and 2 mL of Naphthol AS-MX Phosphate Alkaline (Sigma-Aldrich, St. Louis, MO, USA). Then, cells were washed with PBS and incubated for 30 min with Mayer's Hematoxylin (Sigma-Aldrich, St. Louis, MO, USA). The stained cells were washed and imaged under microscope. Purple staining indicated the synthesis of ALP by osteoblasts.
3
Multiparametric Characterization of BM-hMSC Differentiation
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Immunophenotype analysis was performed by multiparameter flow cytometry before and after the BM-hMSC expansion process using a previously developed protocol [17] . Short tandem repeat analysis was completed by LGC Standards (UK) under their cell line authentication program. Morphology images were obtained using a light microscope (Nikon Eclipse TS-100).
The BM-hMSC differentiation was induced using PRIME-XV Differentiation Serum-Free Medium (Irvine Scientific) according to the manufacturer's instructions. After 21 days the differentiation media were removed, cells rinsed with PBS then fixed with 4% (v/v) paraformaldehyde at room temperature. Adipocytes were stained with 1% (w/v) oil red O (Sigma-Aldrich) in isopropanol at room temperature and rinsed with distilled water. Osteoblasts were incubated with 2.5% (v/v) silver nitrate (Sigma-Aldrich) under ultraviolet light (30-min exposure), rinsed with distilled water and stained with fast violet solution (Sigma-Aldrich) containing 4% (v/v) naphthol AS-MX phosphate alkaline (Sigma-Aldrich) for 45 min at room temperature in the dark. Chondrocytes were stained with 1% (w/v) Alcian blue (Sigma-Aldrich) in 0.1 mol/L hydrochloric acid (Sigma-Aldrich). After 30-min incubation, cells were rinsed three times with 0.1 mol/L HCl. After staining, differentiated cells were visualized under a light microscope (Nikon Eclipse TS-100) [13] .
The BM-hMSC differentiation was induced using PRIME-XV Differentiation Serum-Free Medium (Irvine Scientific) according to the manufacturer's instructions. After 21 days the differentiation media were removed, cells rinsed with PBS then fixed with 4% (v/v) paraformaldehyde at room temperature. Adipocytes were stained with 1% (w/v) oil red O (Sigma-Aldrich) in isopropanol at room temperature and rinsed with distilled water. Osteoblasts were incubated with 2.5% (v/v) silver nitrate (Sigma-Aldrich) under ultraviolet light (30-min exposure), rinsed with distilled water and stained with fast violet solution (Sigma-Aldrich) containing 4% (v/v) naphthol AS-MX phosphate alkaline (Sigma-Aldrich) for 45 min at room temperature in the dark. Chondrocytes were stained with 1% (w/v) Alcian blue (Sigma-Aldrich) in 0.1 mol/L hydrochloric acid (Sigma-Aldrich). After 30-min incubation, cells were rinsed three times with 0.1 mol/L HCl. After staining, differentiated cells were visualized under a light microscope (Nikon Eclipse TS-100) [13] .
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