Alkaline phosphatase (ALP) bioactivity was examined by ALP staining and ALP activity testing after 7 days. For ALP staining, the acid-etched Ti plates loaded with cells and exosome samples were fixed in 4% paraformaldehyde and stained using a BCIP/NBT ALP color development kit (Biyuntian, China), then washed with PBS and photographed. ALP activity was determined using the LabAssay ALP kit (Wako Pure Chemical Industries, Japan) according to the manufacturer’s instructions. Total cellular proteins were determined using the BCA protein assay and the absorbance at 405 nm was measured with a Microplate reader (Thermo Fisher Scientific).
Bcip nbt alp color development kit
Manufactured by Beyotime
Sourced in China
The BCIP/NBT ALP color development kit is a reagent system used to visualize and detect the presence of alkaline phosphatase (ALP) in various biological applications, such as Western blotting, ELISA, and histochemical staining. The kit provides the necessary components to facilitate a colorimetric reaction, resulting in the development of a dark blue-purple precipitate at the sites of ALP activity.
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Lab products found in correlation
Alp assay kit, by Beyotime (14 mentions)
Alkaline phosphatase assay kit, by Beyotime (6 mentions)
Oil red o, by Merck Group (5 mentions)
Alp activity kit, by Nanjing Jiancheng (5 mentions)
Dexamethasone (dex), by Merck Group (5 mentions)
Alp assay kit, by Nanjing Jiancheng (5 mentions)
β glycerophosphate, by Merck Group (5 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Alizarin red s, by Merck Group (3 mentions)
Mesencult adipogenic differentiation kit, by STEMCELL (3 mentions)
Ascorbic acid, by Merck Group (3 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (3 mentions)
Elx808, by Agilent Technologies (3 mentions)
Triton x 100, by Merck Group (2 mentions)
Bca protein quantitation kit, by Beyotime (2 mentions)
Digital camera, by Canon (2 mentions)
Enhanced bca protein assay kit, by Beyotime (2 mentions)
Alp activity assay, by Beyotime (2 mentions)
Microplate reader, by Agilent Technologies (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
96 protocols using bcip nbt alp color development kit
1
ALP Bioactivity Assessment in Osteogenic Cells
2
Colorimetric ALP Detection Protocol
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The expression of ALP was detected using a BCIP/NBT ALP color development kit (Bi Yuntian, Shanghai, China) according to the manufacturer's protocol.
3
Evaluating Osteogenic Differentiation of hBMMSCs
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To evaluate the effect of sample extracts on the early osteogenic differentiation marker ALP, human bone marrow mesenchymal stem-cells (hBMMSCs) were cultured in 12-well plates with sample extracts for 3 and 7 days. The cells were fixed in 4% paraformaldehyde and stained using the BCIP/NBT ALP Color Development Kit (Beyotime, China) for qualitative imaging. Pictures of each well were acquired using an optical microscope (BX51, Olympus, Japan). For the quantification of ALP activity, cells were rinsed with ice-cold PBS and then lysed in 1% Triton X-100 (Sigma, USA) for 10 min on ice. The cells were treated on ice with ultrasound and centrifuged at 12,000 g for 30 min at 4 °C. The protein concentration was measured by correlating the absorbance to protein concentration at 562 nm using a pre-plotted bovine albumin standard curve. The ALP activity was tested using the colorimetric production of p-nitrophenol via p-nitrophenyl phosphate/endogenous ALP enzymatic reaction (Jiancheng, Nanjing, China). Finally, ALP activity was normalized against the total intracellular protein content according to our previous study [25 (link)].
4
Graphene Oxide Quantum Dots Modulate Osteogenic Differentiation
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ALP
is an early marker for the detection of the osteogenic differentiation
of BMSCs. BMSCs were seeded in a 24-well plate at a density of 2 ×
104 cells per well and cultured with an osteogenic-inducing
medium (OIM), then treated with GOQDs of different concentrations
(0, 0.1, 1, 5, and 10 μg/mL). After 7 days of cell culture,
ALP staining was detected by the BCIP/NBT ALP Color Development Kit
(Beyotime, China) according to the manufacturer’s protocol.
For the testing of ALP activity, after 7 days of cell culture, protein
concentrations were detected by the BCA protein test kit (Beyotime,
China), and the ALP activity of BMSCs was detected by the ALP Kit
(Nanjing Jiancheng, China).
is an early marker for the detection of the osteogenic differentiation
of BMSCs. BMSCs were seeded in a 24-well plate at a density of 2 ×
104 cells per well and cultured with an osteogenic-inducing
medium (OIM), then treated with GOQDs of different concentrations
(0, 0.1, 1, 5, and 10 μg/mL). After 7 days of cell culture,
ALP staining was detected by the BCIP/NBT ALP Color Development Kit
(Beyotime, China) according to the manufacturer’s protocol.
For the testing of ALP activity, after 7 days of cell culture, protein
concentrations were detected by the BCA protein test kit (Beyotime,
China), and the ALP activity of BMSCs was detected by the ALP Kit
(Nanjing Jiancheng, China).
5
ALP Expression in C3H10T1/2 Cells
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C3H10T1/2 cells (2 × 104 cells/well) were seeded in 12-well culture plates for 1 day and then the culture medium were replaced by the various extracts. After further cultured for 3 and 7 days, the expression of ALP was stained using the BCIP/NBT ALP color development kit (Beyotime, China) and the intracellular ALP activities were quantitatively assayed using ALP Assay Kit (Beyotime Biotechnology, China). Total protein was measured by BCA protein quantitation kit (Beyotime, China). The ALP activity was normalized with total protein content.
6
Isolation and Differentiation of Mouse Primary BMSCs
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Mouse primary BMSCs were isolated and cultured as described previously.70 (link) To evaluate BMSC proliferation, the number of attached cells was assayed by the Cell Counting Kit-8 (Beyotime) assay according to the manufacturer’s instructions. The optical density at 450 nm was determined with a microplate reader (PerkinElmer). For osteogenic differentiation, BMSCs were cultured in osteogenic medium (α-MEM containing 10% FBS and 50 μg·mL−1 ascorbic acid) for 7 days and then stained for ALP using a BCIP/NBT Alp color development kit (Beyotime, China) or cultured in osteogenic medium for 7 days followed by mineralization-inducing medium (osteogenic medium plus 2.5 mmol·L−1 β-glycerophosphate for 7 days and then subjected to alizarin red S (40 mmol·L−1, pH 4.2) (Sigma) staining. For adipogenic differentiation, BMSCs were cultured with reagents from the MesenCult™ Adipogenic Differentiation Kit (STEMCELL Technologies) for 9 days and then stained with Oil Red O (Sigma).
7
Isolation and Differentiation of Mouse BMSCs
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Mouse primary BMSCs were isolated from both femurs and tibiae and cultured as described previously [43 (link)]. For osteogenic differentiation, BMSCs were cultured in osteogenic medium (α-MEM containing 10% FBS and 50 μg/mL ascorbic acid) for 7 days and then stained for ALP using a BCIP/NBT ALP color development kit (Beyotime, China). BMSCs were differentiated in osteogenic medium for 14 days for alizarin red S staining using Alizarin Red S Staining Kit (Beyotime, China) and qPCR analysis. For adipogenic differentiation, BMSCs were cultured with reagents from the MesenCult™ Adipogenic Differentiation Kit (Stemcell Technologies) for 9 days and then stained with Oil red O (Sigma) and qPCR analysis.
8
ALP and Mineralized Nodule Staining Protocols
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A 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) ALP color development kit (Beyotime) was used to stain the ALP activity of hBMSCs. After hBMSCs were fixed in 4% paraformaldehyde for 30 min, pre-prepared BCIP/NBT dyeing was added and incubated with samples for 1 h to stain ALP. BCIP/NBT dyeing was removed and rinsed by distilled water to terminate the staining reaction. The images of ALP staining were captured by a digital camera (Canon) and microscopy (Olympus).
ARS staining was used to measure the property of mineralized nodule formation. Briefly, BMSCs were fixed in 75% ethanol for 1 h. Afterward, 2% ARS solution (pH 4.2, Sigma–Aldrich) was dripped onto the samples and incubated for 10 min. Unreacted ARS was rinsed thoroughly using distilled water. The images of deposited calcium were captured by a digital camera (Canon) and microscopy (Olympus).
ARS staining was used to measure the property of mineralized nodule formation. Briefly, BMSCs were fixed in 75% ethanol for 1 h. Afterward, 2% ARS solution (pH 4.2, Sigma–Aldrich) was dripped onto the samples and incubated for 10 min. Unreacted ARS was rinsed thoroughly using distilled water. The images of deposited calcium were captured by a digital camera (Canon) and microscopy (Olympus).
9
ALP Activity Quantification in BMSCs
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A 5-bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium (BCIP/NBT) ALP color development kit (Beyotime) and ALP assay kit (Beyotime) was used to stain and quantify the ALP activity of BMSCs, respectively. After BMSCs were fixed in 4% paraformaldehyde for 30 min, pre-prepared BCIP/NBT dyeing was added and incubated with samples for 1 h to stain ALP. BCIP/NBT dyeing was removed and rinsed by distilled water to terminate the staining reaction. The images of ALP staining were captured by a digital camera (Canon). For ALP quantification, BMSCs were lysed by using 1% v/v Triton X-100 (Biofroxx) on ice for 30 min. The cell-lysis solution was centrifuged for 30 min at 12000 rpm under 4 °C and then the supernatant was collected. Afterward, 50 μL supernatant was mixed with equal volume ALP assay working solution and incubated for another 30 min. The ALP activity was measured at 405 nm wavelength by using an enzyme-labeling instrument (BioTech).
10
Evaluating Early Osteogenic Differentiation of BMSCs
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To evaluate the early markers of osteogenic differentiation of BMSCs, ALP staining and ALP activity determination were performed on days 7 and 14. Briefly, after washing gently, cells–scaffolds were incubated in BCIP/NBT ALP Color Development Kit (Beyotime, Shanghai, China) solution for 30 min at room temperature in darkness. Subsequently, samples were photographed by an optical microscope (Eclipse Ci Pol, Nikon) to visualize the ALP activity.
Additionally, an ALP Assay Kit (Beyotime) was used for quantitative determination of ALP activity. In brief, cells–scaffolds were washed twice, lysed with a cell lysis buffer, and centrifuged at 4°C at 6000 rpm for 10 min. Then the supernatant was extracted, para-nitrophenyl phosphate (pNPP) substrate was added and incubated at 37°C for 30 min. Meanwhile, BCA Protein Assay Kit (Beyotime) was used to measure total protein in supernatant. The absorbance of the resultant yellow and purple compound were determined at 405 and 562 nm with a microplate reader, respectively. The measured ALP concentration for each sample was then normalized to total protein concentration.
Additionally, an ALP Assay Kit (Beyotime) was used for quantitative determination of ALP activity. In brief, cells–scaffolds were washed twice, lysed with a cell lysis buffer, and centrifuged at 4°C at 6000 rpm for 10 min. Then the supernatant was extracted, para-nitrophenyl phosphate (pNPP) substrate was added and incubated at 37°C for 30 min. Meanwhile, BCA Protein Assay Kit (Beyotime) was used to measure total protein in supernatant. The absorbance of the resultant yellow and purple compound were determined at 405 and 562 nm with a microplate reader, respectively. The measured ALP concentration for each sample was then normalized to total protein concentration.
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