ALP staining was performed following 7 days osteogenic stimulation. Briefly, cells were rinsed twice with PBS and fixed with 4% formaldehyde for 30 min at room temperature. The fixed cells were stained with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium solution (Beyotime Institute of Biotechnology, Shanghai, China) for 30 min at room temperature and washed 3 times with PBS. ARS staining was used to assess mineral deposition produced at late-stage bone formation. Briefly, following 14 days osteogenic stimulation, cells were washed twice with PBS, fixed with 4% formaldehyde for 30 min at room temperature, washed twice with PBS and then stained with the Alizarin Red S Solution (Cyagen Biosciences, Inc., Chicago, USA) for 30 min at room temperature. Following ARS staining, the cells were washed 3 times with PBS. Images were captured using an Olympus SZX12 inverted microscope equipped with a digital camera and connected to a PC using MagnaFire 2.0 camera software.
5 bromo 4 chloro 3 indolyl phosphate nitroblue tetrazolium solution
Manufactured by Beyotime
Sourced in China
5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium solution is a chromogenic substrate used for the detection and visualization of enzymatic activity, particularly alkaline phosphatase. The solution contains 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium as the active components.
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2 protocols using 5 bromo 4 chloro 3 indolyl phosphate nitroblue tetrazolium solution
1
Osteogenic Differentiation Assays
2
Alkaline Phosphatase Activity in hDPSCs
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hDPSCs were cultured with 1 μg/mL LPS, 1× CGF, or a combination of LPS and CGF for 4 and 7 days. The culture medium was replaced with fresh culture medium every 2 days. For ALP staining, the media were removed, and the cells were fixed in 70% ethanol for 1 h. After the cells were rinsed three times with deionized water, a 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium solution (Beyotime, Shanghai, China) was added to each well. Then, the stained cells were photographed after several steps of washing. For quantitative analysis, the stain was extracted with 10% (w/v) cetylpyridinium chloride (Sigma-Aldrich) for 15 min, and stain intensity was quantified by measuring the absorbance at 562 nm on an absorbance microplate reader (BioTek, Winooski, VT, USA).
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