An anthraquinone dye assay was used to assess osteogenic differentiation on days 7 and 14 in order to gauge the calcium deposits and the level of alkaline phosphatase activity [20 (link)]. Cell spheroids grown on culture plates containing osteogenic magnesium were obtained on days 7 and 14. Using a commercial kit, alkaline phosphatase activity was assessed (K412-500, BioVision, Inc., Milpitas, CA, USA). In order to measure the absorbance at 405 nm, cell lysates were added to an assay solution (K412-500; BioVision, Inc.) together with a 5 mM p-nitrophenylphosphate substrate [19 (link)]. The combination was then incubated at 40 °C for 30 min. After being cleaned, fixed, and stained for 30 min at room temperature with a 2% Alizarin Red S solution (cat. no. 0223; ScienCell Research Laboratories, Inc.), stem cell spheroids were processed. The bound dyes were then measured for 15 min at 560 nm with 10% cetylpyridinium chloride (cat. no. C0732; Sigma-Aldrich; Merck KGaA, Saint Louis, MO, USA) [21 (link)].
K412 500
Manufactured by Abcam
Sourced in United States, Germany
K412-500 is a lab equipment product. It is a 500 ml bottle containing a reagent or solution for use in laboratory procedures. The core function of this product is to provide the specified reagent or solution required for certain laboratory applications. However, without more details about the specific contents and intended use, a detailed description cannot be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Agilent Technologies (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Stempro osteogenesis differentiation kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Cetylpyridinium chloride, by Merck Group (1 mentions)
Alizarin red s solution, by ScienCell (1 mentions)
Ckx41sf, by Olympus (1 mentions)
Lysis buffer, by Merck Group (1 mentions)
Radioimmunoprecipitation assay (ripa), by Merck Group (1 mentions)
Ab113344, by Abcam (1 mentions)
Ab208348, by Abcam (1 mentions)
Mop00, by R&D Systems (1 mentions)
Mtr00, by R&D Systems (1 mentions)
Phosphate buffered saline (pbs), by Welgene (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Ascorbic acid 2 phosphate, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
23 protocols using k412 500
1
Quantifying Osteogenic Differentiation
2
Osteogenic Differentiation Evaluation
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On days 7 and 14, osteogenic differentiation was evaluated using an anthraquinone dye assay to measure calcium deposits and the degree of alkaline phosphatase activity [17 (link)]. On days 7 and 14, cell spheroids cultivated on culture plates with an osteogenic medium were obtained. Alkaline phosphatase activity was measured using a commercial kit (K412-500, BioVision, Inc., Milpitas, CA, USA). After combining the cell lysates with an assay solution (K412-500; BioVision, Inc.) and incubating it at 4 °C for 30 min, the absorbance at 405 nm was measured [16 (link)].
3
Osteogenic Differentiation Evaluation
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The level of alkaline phosphatase activity and an anthraquinone dye assay for calcium deposit evaluation were used to assess osteogenic differentiation (16 (link)). We obtained cell spheroids grown on culture plates with osteogenic media on Days 3, 7, 14, and 21. We used a commercially available kit (K412-500, BioVision, Inc.) to evaluate level of alkaline phosphatase activity.
We used an anthraquinone dye assay for calcium deposit evaluation to assess osteogenic differentiation on Days 7, 14, and 21(17 (link)). We washed three times with phosphate-buffered saline and then fixed with 4% paraformaldehyde in phosphate-buffered saline at room temperature for 15 min. After that, we carefully removed the fixative and washed three times with deionized water. We added 2% Alizarin Red S Staining solution, and then incubated for 20 min. We removed dye and washed three times with deionized water. The quantification of the bound dyes was performed afterwards by adding 10% cetylpyridinium chloride for 15 min at ambient temperature. Spectrophotometric quantification was performed at 560 nm.
We used an anthraquinone dye assay for calcium deposit evaluation to assess osteogenic differentiation on Days 7, 14, and 21(17 (link)). We washed three times with phosphate-buffered saline and then fixed with 4% paraformaldehyde in phosphate-buffered saline at room temperature for 15 min. After that, we carefully removed the fixative and washed three times with deionized water. We added 2% Alizarin Red S Staining solution, and then incubated for 20 min. We removed dye and washed three times with deionized water. The quantification of the bound dyes was performed afterwards by adding 10% cetylpyridinium chloride for 15 min at ambient temperature. Spectrophotometric quantification was performed at 560 nm.
4
Evaluating ALP Activity in Osteo-induced hBM-MSCs
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ALP activity was evaluated in osteo-induced hBM-MSCs treated with or without samples. Cells were seeded in 6-well plates (1 × 106 cells/well) and osteo-induced as previously described. At day 7 of differentiation, cells were lysed with 0.1% Triton X-100 and 25 mM carbonate buffer after washing with PBS. The cellular ALP activity was assessed using the supernatants of the cell lysates following 15 min centrifugation at 4 °C (12,000× g). The total protein content of the supernatant was determined by the Bradford protein determination method. Analysis of ALP activity was performed using a commercial kit (K412-500; BioVision, Hannover, Germany) according to the producer’s instructions.
5
Alkaline Phosphatase and Alizarin Red Assays
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We performed the alkaline phosphatase activity assays using a kit (K412-500, BioVision, Inc., Milpitas, CA, USA) on Days 1, 5, and 7 following the manufacturer’s protocol. In short, the cell spheroids were suspended in an assay buffer, sonicated, and centrifuged. Then the supernatant was mixed with a p-nitrophenylphosphate substrate and incubation was performed. The optical density of the resultant p-nitrophenol was measured spectrophotometrically at 405 nm. We performed Alizarin Red S staining on Days 7 and 14.
6
Osteogenic Differentiation of Stem Cells
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To access the osteogenic differentiation of stem cells, absorbance at 405 nm was measured using an alkaline phosphatase assay kit (K412-500, BioVision, Inc., Milpitas, CA, USA) according to the manufacturer’s protocol after 1, 3, 7 and 14 days of cell culture using a microplate reader (BioTek Instruments Inc.).
The cells were washed, fixed and colored with 2% Alizarin Red S Solution (ScienCell Research Laboratories, Inc., Carlsbad, CA, USA) after 7 and 14 days of cell culture. The stained cells were visualized using a microscope (CKX41SF, Olympus Corporation). Ten percent cetylpyridinium chloride (Sigma-Aldrich Co.) was used to dissolve the bound dye and quantification was performed spectrophotometrically at 560 nm.
The cells were washed, fixed and colored with 2% Alizarin Red S Solution (ScienCell Research Laboratories, Inc., Carlsbad, CA, USA) after 7 and 14 days of cell culture. The stained cells were visualized using a microscope (CKX41SF, Olympus Corporation). Ten percent cetylpyridinium chloride (Sigma-Aldrich Co.) was used to dissolve the bound dye and quantification was performed spectrophotometrically at 560 nm.
7
Osteogenic Differentiation and ALP Assay
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Cell spheroids grown in osteogenic induction media (STEMPRO Osteogenesis Differentiation kit; Gibco; Thermo Fisher Scientific, Inc.) at 37°C were obtained on days 1 and 5. Alkaline phosphatase activity assays were conducted using a commercially available kit (K412-500; BioVision, Inc., Milpitas, CA, USA) at days 1 and 5, according to the manufacturer's instructions. The cells were resuspended in assay buffer, sonicated and subsequently centrifuged at 13,000 g for 3 min at 4°C to remove insoluble material. Supernatant was mixed with p-nitrophenylphosphate substrate and incubated at 25°C for 60 min. The optical density of the resultant p-nitrophenol at 405 nm was determined spectrophotometrically.
8
Osteogenic Differentiation Assessment
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The assessment of osteogenic differentiation involved analyzing alkaline phosphatase activity levels and using an anthraquinone dye assay to evaluate calcium deposition [20 (link)]. Cell spheroids generated in culture wells with osteogenic medium were collected on Days 7 and 14. The assessment of alkaline phosphatase activity was conducted using a commercial kit (K412-500, BioVision, Inc., Milpitas, CA, USA).
On Days 7 and 14, we evaluated calcium deposits using an anthraquinone dye assay to determine osteogenic differentiation [21 (link)]. After washing and fixing the stem cell spheroids, we dyed them with Alizarin Red S at room temperature for 30 min. Following the extraction, the bound dyes were quantified using cetylpyridinium chloride.
On Days 7 and 14, we evaluated calcium deposits using an anthraquinone dye assay to determine osteogenic differentiation [21 (link)]. After washing and fixing the stem cell spheroids, we dyed them with Alizarin Red S at room temperature for 30 min. Following the extraction, the bound dyes were quantified using cetylpyridinium chloride.
9
Quantifying Osteogenic Potential via ALP Assay
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The osteogenic potential was assessed using an ALP assay kit (BioVision K412-500) after 7 days. The cells were washed twice with ice-cold PBS and lysed using 300 μL of RIPA (radioimmunoprecipitation assay) lysis buffer (Sigma-Aldrich Corp., St Louis, MO, USA) for 5 mins on ice. The cells were then rapidly scraped from the plate, and the cell lysates/RIPA buffer were transferred to a 1.5-mL microcentrifuge tube on ice for 20 min, followed by centrifugation at 8000 g for 10 min at 4°C. The supernatant was then added to a new 1.5-mL microcentrifuge tube and stored at -20°C. 50 μL of 5 mM pNPP solution was added to each well containing the test samples and the aliquots were incubated for 60 min at 25°C while protected from light. Subsequently, 20 μL of the stop solution was added to terminate the ALP activity in the sample. The absorbance at 405 nm was measured with a spectrophotometer (UV-Vis 8500). Then, the ALP activity was calculated using the following formula: ((optical density–mean optical density of the control wells) × total volume × dilution)/(18.45 × sample volume).
10
Mouse Serum Biomarker Profiling
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The following bone markers and proinflammatory cytokines in mouse serum were measured using commercially available kits according to the manufacturer’s manual instructions: alkaline phosphatase (ALP) (Catalog #K412–500, BioVision, Milpitas, CA, USA), osteocalcin (OC) (Catalog #SEA471Mu, USCN Life Science Inc., Wuhan, China), cross-linked C-telopeptide of type I collagen (CTX1) (Catalog #CEA665Mu, USCN Life Science Inc.), osteoprotegerin (OPG) (Catalog #MOP00, R&D Systems Inc., Minneapolis, MN, USA), receptor activator of nuclear factor κ-B ligand (RANKL) (Catalog #MTR00, R&D Systems Inc.), interleukin-1α (IL-1α) (Catalog #ab113344, Abcam, Cambridge, MA, USA), IL-1β (Catalog #ab108866, Abcam), IL-6 (Catalog #ab100712, Abcam) and tumor necrosis factor-α (TNF-α) (Catalog #ab208348, Abcam). The measurements were conducted by an automated microplate reader (Tu et al., 2021 (link)).
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