Cells at a density of 1×105 cells/well were cultured with samples in 24-well plates.After 24 hrs of culture, the culture medium was replaced with osteogenic induction medium containing serum and then changed every 3 days. At days 7, 10, and 14, 200 μL Nonidet P40 solution (1%) was added and incubated for another 1 hr to obtain the cell lysate. Then, the cell lystae was centrifuged and 50 μL supernatant transferred into a new 96-well plate, and 50 μL of p-nitrophenylphosphate (2 mg/mL, Sangon, Shanghai, China) solution consisting of glycine (0.1 mol/L), MgCl2·6H2O (1 mmol/L) was added for another 30 mins. Subsequently, this reaction was stopped by adding 100 μL NaOH (0.1 mol/L) solution. ALP absorbance was quantified using microplate reader at a wavelength of 405 nm. The total protein content in cell lysate was determined using the bicinchoninic acid method in aliquots of the same samples with a Pierce protein assay kit (Pierce Biotechnology Inc., Rockford, Illinois, USA), read at 562 nm, and calculated according to a series of BSA standards. ALP activity was normalized to total protein content and was expressed as absorbance at OD405/total protein.
P nitrophenylphosphate
Manufactured by Sangon
Sourced in China
P-nitrophenylphosphate is a substrate used in biochemical assays to detect and measure the activity of phosphatase enzymes. It serves as a colorimetric reagent, producing a yellow compound upon enzymatic hydrolysis.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax 384, by Molecular Devices (3 mentions)
Pierce protein assay kit, by Thermo Fisher Scientific (1 mentions)
Multimode microplate reader, by Agilent Technologies (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Synergy h1 hybrid, by Agilent Technologies (1 mentions)
Mgcl2, by Sangon (1 mentions)
6 protocols using p nitrophenylphosphate
1
Osteogenic Differentiation Assay
2
SEAP Quantification in Cell Culture
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The quantification of SEAP production in culture supernatant was conducted as previously reported [38 (link)]. In brief, the substrate solution was configured with 2 × assay buffer (containing 1 mM MgCl2, 21% (w/w) diethanolamine, 20 mM homoarginine (catalog. no. 1483-01-8, Sangon Biotech, Inc.) (pH 9.8)), and substrate (containing 120 mM p-nitro phenyl phosphate (Catalog. no. 333338-18-4, Sangon Biotech, Inc.)) at a ratio of 5 : 1 (v/v). Then, 120 μL of substrate solution was added to 80 μL heat-inactivated (incubation for 30 min at 65°C) culture medium, and the 405 nm absorption was measured using multimode microplate reader (BioTek Instruments, Inc., USA). Blood SEAP in mice was measured using a chemiluminescence-based assay kit (catalog no. 11779842001, Roche Diagnostics GmbH, Inc.).
3
Osteogenic Differentiation of rBMSCs
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For osteogenic differentiation of rBMSCs, cells were cultured in osteogenic medium (basal medium additionally provided with 10 mM β-glycerophosphate, 50 μg/mL ascorbic acid and 100 nM dexamethasone). And rBMSCs were seeded on the scaffold samples and alkaline phosphatase (ALP) activity of cells was measured at 1, 4 and 7 days to evaluate the effect of Cu-doped n-CDHA/MAC on the early osteogenic differentiation. Medium was changed up every two days. Briefly, we aspirated the culture medium, added 200 μl of 1% Nonidet P-40 (NP-40) solution to each well and incubated for 1 h at 37 °C. After the cell lysate was centrifuged, 50 μl supernatant was added to 96-well plates. 50 μl, 2 mg/ml P-nitrophenyl phosphate (Sangon, Shanghai, China) substrate solution including 0.1 mol/L glycine and 1 mmol/L MgCl2·6H2O was added and incubated for 30 min at 37 °C. At last, we added 100 μl 0.1 N NaOH to quench the reaction. The absorbance of ALP at λ=405 nm was quantified on a microplate reader (SPECTRAmax 384, Molecular Devices, USA). The total protein content was measured by the bicinchoninic acid protein assay kit (Pierce Biotechnology Inc, Rockford, IL). The absorbance was read at λ=405 nm and the total protein was estimated. The ALP levels were obtained as the changed OD values and normalized to the total protein content.
4
Osteogenic Differentiation of MT3T3-E1 on Scaffolds
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MT3T3-E1 cells were seeded on the scaffolds (WG, WMC20 and WMC40), and then incubated with the osteogenic induction medium in 24-well plates. At day 7, 10 and 14, 200 μL 1% Nonidet P-40 (NP-40) solution was added into each well after the culture medium aspirated, and incubated for 1 h. The cell lysate was obtained and centrifuged. Exact 50 mL of supernatant was added to 96-well plates, 50 μL of 2 mg/mL p-nitrophenyl-phosphate (Sangon, Shanghai, China) substrate solution composed of 0.1 mol L−1 glycine, 1 mmol L−1 MgCl2·6H2O was added and incubated for 30 min at 37 °C. The reaction was quenching by adding 100 μL of 0.1 N NaOH, and the absorbance of ALP was quantified at a wavelength of 405 nm using a microplate reader (SPECTRAmax 384, Molecular Devices, USA). ALP activity was normalized to protein quantity of scaffolds. ALP activity was normalized to protein quantity of scaffolds, and all the experiments were performed for 3 times.
5
Quantification of Placental SEAP Secretion
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The expression of human placental SEAP in a cell culture medium was quantified using a p-nitrophenyl phosphate-based light absorbance time course assay. Briefly, 120 µL of substrate solution [100 µL of 2 × SEAP buffer (pH 9.8) containing 20 mM L-homoarginine hydrochloride (catalog no. A602842, Sangon Biotech), 1 mM MgCl2 (catalog no. A610328, Sangon Biotech), and 21% (v/v) diethanolamine (catalog no. A600162, Sangon Biotech), and 20 µL of substrate solution containing 120 mM p-nitrophenyl phosphate (catalog no. 333338-18-4, Sangon Biotech)] were added to 80 µL of heat-inactivated (65 °C, 30 min) cell culture supernatant69 (link). The time course of absorbance at 405 nm was measured at 37 °C using a Synergy H1 hybrid multimode microplate reader (BioTek Instruments) with Gen5 software (version 2.04).
6
ALP Activity Assay for Cell Differentiation
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For cell differentiation assay, MG3T3 cells were seeded onto the different CSi-Mgx ceramic scaffolds in the 24-well plates and incubated for 14 d, with those cultured in the scaffold-free condition as control. After the culture medium was aspirated, each well was added into with 200 μL of 1% Nonidet P-40 (NP-40) solution and incubated at room temperature for 1 h. The cell lysate was centrifuged and 50 μL of supernatant was transferred to 96-well plates. Fifty milliliters of p-nitrophenylphosphate (Sangon, Shanghai, China) substrate solution (2 mg/mL) composed of 0.1 mol l−1 glycine, 1 mmol l−1 MgCl. 6H2O was added to the 96-well plates and incubated for 30 min at 37 °C. The reaction was quenched by adding 100 mL of 0.1 N NaOH. The absorbance of ALP was quantified at a wavelength of 405 nm using a microplate reader (SPECTRAmax 384, Molecular Devices, USA). The total protein content in the cell lysate was determined using the bicinchoninic acid method in aliquots of the same samples with the Pierce protein assay kit (Pierce Biotechnology Inc., Rockford, IL, USA), read at 562 nm and calculated according to a series of albumin (bovine serum albumin) standards. The ALP levels were normalized to the total protein content, and all the experiments were performed in quadruple.
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