Cells were seeded on the membrane (15 mm in diameter) with a density of 1.5 × 105 cells/mL in 24-well plates, and ALP staining was performed by using the ALP staining kit (Beyotime, Suzhou, China) at 1 and 7 days. The quantitative ALP activity was determined using p-nitrophenyl phosphate (pNPP; Beyotime, Suzhou, China). The absorbance values (OD) were recorded at a wavelength of 405 nm to determine the ALP activity. The total protein content was assessed by using a BCA protein assay kit (Sigma, USA), and the OD values were normalized to the bovine serum albumin (BSA; Sigma, USA) standard curve at 590 nm. Then, the level of pNPP production was assessed at the OD value of 405 nm. After osteogenic induction in the osteogenic medium for 21 days, the mineral deposition by cultured BMSCs was assessed by Alizarin Red S staining: The cells were washed thrice with PBS, fixed in 70% ethanol for 30 min, and stained with Alizarin Red S (ARS; Sigma, USA) for 30 min at 37 °C.
P nitrophenyl phosphate pnpp
Manufactured by Beyotime
Sourced in China
P-nitrophenyl phosphate (pNPP) is a colorless, water-soluble organic compound used as a substrate in various biochemical and analytical applications. It is commonly used as a colorimetric reagent to measure the activity of enzymes, particularly phosphatases, by monitoring the release of the yellow p-nitrophenol product upon enzymatic hydrolysis.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Bca protein assay kit, by Merck Group (2 mentions)
Alizarin red s, by Merck Group (1 mentions)
Alp staining kit, by Beyotime (1 mentions)
Alizarin red s ar, by Merck Group (1 mentions)
Eclipse 100, by Nikon (1 mentions)
Bcip nbt alkaline phosphatase color development kit, by Beyotime (1 mentions)
Cetylpyridinium chloride, by Merck Group (1 mentions)
Bcip nbt solution, by Beyotime (1 mentions)
Alizarin red s solution, by Beyotime (1 mentions)
Microplate reader, by Thermo Fisher Scientific (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
5 protocols using p nitrophenyl phosphate pnpp
1
Quantitative Evaluation of Osteogenic Differentiation
2
Quantifying Osteogenic Differentiation of BMSCs
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Quantitative ALP activity and staining assays were performed at day 7 after BMSCs were treated with icariin at concentrations of 0, 10, 20 and 40 μM. Samples from all groups were incubated with p-nitrophenyl phosphate (pNPP) (Beyotime, Suzhou, China) at 37 °C for 30 min. The absorbance values (OD) were recorded at 405 nm to determine ALP activity. Total protein content was assessed using a BCA protein assay kit (Sigma, USA), and the OD values were normalized to bovine serum albumin (BSA, Sigma, USA) standard curve at 590 nm. ALP activity was assessed as the OD value at 405 nm per milligram of total protein. In addition, ALP staining was performed according to the manufacturer’s instructions (Beyotime, Suzhou, China) and was then observed by a digital camera (Eclipse-100, Nikon, Tokyo, Japan). All experiments were performed in triplicate.
3
Alkaline Phosphatase and Mineralization Assays
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After 7 days of inoculation on the surfaces of the different samples, cells (100 μL, 4 × 104 cells/mL) were fixed using 4% PFA, and a BCIP/NBT alkaline phosphatase color development kit (Beyotime, Shanghai, China) was added for staining for 20 h at 37 °C. The samples were rinsed twice with deionized water, dried, and photographed under a microscope. For the quantitative ALP assay, the cell lysates were incubated with p-nitrophenyl phosphate (pNPP, Beyotime, Shanghai, China) at 37 °C for 30 min. ALP activity was tested by detecting the optical density (OD) values at 405 nm.
After 14 days of inoculation on the surfaces of the different samples, the cells (100 μL, 4 × 104 cells/mL) on the membranes were fixed using 4% PFA and stained using 0.1% alizarin red S (ARS). The samples were rinsed twice with deionized water, dried and photographed. For the quantitative assay, the stained cells were desorbed with 10% cetylpyridinium chloride (Sigma, St. Louis, MO, USA), and the OD values were detected at 562 nm.
After 14 days of inoculation on the surfaces of the different samples, the cells (100 μL, 4 × 104 cells/mL) on the membranes were fixed using 4% PFA and stained using 0.1% alizarin red S (ARS). The samples were rinsed twice with deionized water, dried and photographed. For the quantitative assay, the stained cells were desorbed with 10% cetylpyridinium chloride (Sigma, St. Louis, MO, USA), and the OD values were detected at 562 nm.
4
Osteogenic Differentiation of Mouse Stem Cells
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mSSCs were trypsinized and replated in a 12-well plate at a concentration of 5 × 104 cells per well. After being incubated in complete medium for 2 days, the medium was replaced by osteogenic induction medium (Oricell, Shanghai, China). The induction medium was changed every 3 days. The alkaline phosphatase (ALP) assay and alizarin red S (ARS) staining were conducted, respectively, on 14 days and 21 days of osteogenic induction. After being fixed for 30 min at room temperature with 10% neutral-buffered formalin, mSSCs were incubated with a BCIP/NBT solution (Beyotime, Shanghai, China) or alizarin red S solution (Beyotime) at room temperature for 10 min. ALP activities were assessed using p-nitrophenyl phosphate (pNPP, Beyotime), and the OD values were measured at 405 nm. ARS staining were quantified using 10% cetylpyridinium chloride (CPC, Beyotime) in 10 mM sodium phosphate for 15 min at room temperature, and the OD values were measured at 562 nm.
5
Osteoblast ALP Staining and Activity Assay
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Osteoblasts were seeded in 12-well plates at a density of 5×104 cells/well in osteogenic medium for 7 days. ALP staining of osteoblasts were conducted using a commercial staining kit (Beyotime Biotechnology). In brief, osteoblasts were fixed with 4% paraformaldehyde and gently washed with PBS, after which they were stained with NBT/BCIP staining solution at room temperature for 30 min in dark conditions. Representative images were acquired for follow-up comparison.
In ALP activity assay, 1% triton-X was added to lyse osteoblasts, and the supernatant was collected (18 (link)). The relative activity of ALP was analyzed using p-nitrophenyl phosphate (pNPP; Beyotime) as the substrate. The mixture of supernatant and pNPP was incubated at 37 °C for 30 min on a bench shaker and the reaction was stopped by 20 nM NaOH (Sigma-Aldrich). The absorbance was measured at 405 nm using a microplate reader (Thermo Fisher). Finally, ALP activity was normalized by total protein concentration measured by a BCA protein assay kit (Beyotime Biotechnology).
In ALP activity assay, 1% triton-X was added to lyse osteoblasts, and the supernatant was collected (18 (link)). The relative activity of ALP was analyzed using p-nitrophenyl phosphate (pNPP; Beyotime) as the substrate. The mixture of supernatant and pNPP was incubated at 37 °C for 30 min on a bench shaker and the reaction was stopped by 20 nM NaOH (Sigma-Aldrich). The absorbance was measured at 405 nm using a microplate reader (Thermo Fisher). Finally, ALP activity was normalized by total protein concentration measured by a BCA protein assay kit (Beyotime Biotechnology).
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