For osteogenic differentiation assay, MC3T3-E1 cells were seeded at a density of 1.0 × 104 cells per cm2 on PEEK and PEEK/ZnO composites. One day after the cells were seeded, the medium was replaced with a differentiation medium α-DMEM (Gibco) with 10% FBS, 1% penicillin/streptomycin, 50 μg mL−1 ascorbic acid (Sigma Aldrich), 10 mM β-glycerophosphate phosphate (Sigma Aldrich) and 10 nM dexamethasone (Sigma Aldrich). Cells of the eight groups of treatment were collected from each well at different time points and assayed for ALP activity. Each component ALP activity was determined using a commercially available ALP kit (Nanjing Jiancheng Biotech Co., Ltd) according to standard procedures. The 24-well plate (Croning) was washed three times with PBS; then, 200 μL of 0.1% Triton X-100 was added to lyse the cells for 30–40 minutes and subsequently, the reaction reagents were added according to the manufacturer's recommendations. Finally, the ALP absorbance was measured at 520 nm using a microplate reader (Tecan Auatria GmbH). Protein concentration was measured at 595 nm using a protein assay reagent (Beyotime Bradford Protein Assay Kit) according to the manufacturer's instructions, and the level of ALP activity was normalized by the amount of total protein.
β glycerophosphate phosphate
Manufactured by Merck Group
β-glycerophosphate is a chemical compound used as a buffer in laboratory settings. It is a source of phosphate ions and is commonly used to maintain a specific pH range in various biological and biochemical experiments.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (3 mentions)
Ascorbic acid, by Merck Group (3 mentions)
Bradford protein assay kit, by Beyotime (1 mentions)
Alp kit, by Nanjing Jiancheng (1 mentions)
α dmem, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Alizarin red staining, by Merck Group (1 mentions)
Cetylpyridinium chloride, by Merck Group (1 mentions)
Automated microplate reader, by Bio-Rad (1 mentions)
P nitrophenyl phosphate pnpp, by Merck Group (1 mentions)
Radioimmunoprecipitation assay (ripa), by Merck Group (1 mentions)
3 protocols using β glycerophosphate phosphate
1
Osteogenic Differentiation of MC3T3-E1 Cells
2
Alizarin Red Staining of Mineralized Nodules
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Cells were seeded in 24-well plates into 3 repeat wells at a density of 10 5 cells per well with α-MEM mineralizing medium containing 10% FBS, 1% penicillin/streptomycin, 100 μg/mL ascorbic acid (Sigma), 10 mM β-glycerophosphate phosphate (Sigma) and 1 × 10 -8 M dexamethasone (Sigma) at 4 different glucose concentrations with or without LY294002. The mineralized nodule formation was detected at 7 and 14 days using alizarin red staining (Sigma). Briefly, cells were rinsed twice with phosphate-buffered saline (PBS) followed by fixation with 4% paraformaldehyde (Sigma) for 30 minutes at room temperature. Then, cells were stained with 2% alizarin red-S (pH 4.2) for 20 minutes at room temperature and extensively rinsed with distilled water. For quantification, the bound staining was eluted with 10% (wt/vol) cetylpyridinium chloride (Sigma) and the absorbance of supernatants was measured by the automated microplate reader (Bio-Rad) at 570 nm.
3
Osteoblast Differentiation via ALP
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To evaluate osteoblastic differentiation, alkaline phosphatase (ALP) activity was measured at 3 and 7 days. Cells were seeded in 96-well plates (10 4 cells/well) into 5 repeat wells with α-MEM mineralizing medium containing 10% FBS, 1% penicillin/streptomycin, 100 μg/mL ascorbic acid (Sigma), 10 mM β-glycerophosphate phosphate (Sigma) and 1 × 10 -8 M dexamethasone (Sigma) at 4 different glucose concentrations with or without LY294002. At each time point, cells were sonicated in RIPA (Sigma) buffer to obtain the cell lysates. ALP activity was measured using p-nitrophenyl-phosphate (pNPP; Sigma) as substrate in assay buffer (10 mM MgCl 2 and 0.1 M alkaline buffer, pH 10.3). After incubating at 37°C for 30 minutes, the reaction was stopped by the addition of 0.4 M NaOH and the amount of p-nitrophenol released was estimated by measuring the absorbance at 405 nm. Relative ALP activity is defined as millimoles of pNPP hydrolyzed per minute per mg of total protein (units).
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