The protocol for BMSC osteogenic differentiation was described in our previous study.25 Briefly, HG‐treated BMSCs were seeded into 24‐well or 6‐well plates at a density of 1 × 104 cells/cm2 and then cultured in HG‐DMEM (25 mmol/L), and BMSCs cultured in LG‐DMEM (5.5 mmol/L) were considered the control group. After the cells reached 80% confluence, the medium was replaced with osteogenic induction medium (OIM), and 1 nmol/L dexamethasone, 50 μmol/L L‐ascorbic acid‐2‐phosphate and 20 mmol/L β‐glycerophosphate (Cyagen Biosciences, Guangzhou, China) were added to the corresponding culture medium with different glucose concentrations. Morroniside, FPS‐ZM1 and BBGCP2 were added to the OIM with the concentrations mentioned above.
β glycerophosphate
Manufactured by Cyagen
Sourced in China, United States
β-glycerophosphate is a chemical compound used in cell culture media as a source of phosphate. It is a common supplement in osteogenic differentiation protocols for mesenchymal stem cells and osteoblast cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Cyagen (11 mentions)
Insulin, by Cyagen (6 mentions)
Ascorbate, by Cyagen (4 mentions)
Rosiglitazone, by Cyagen (4 mentions)
Glutamine, by Cyagen (4 mentions)
Penicillin streptomycin, by Cyagen (3 mentions)
L ascorbic acid 2 phosphate, by Cyagen (2 mentions)
Alizarin red s, by Cyagen (2 mentions)
Indomethacin, by Cyagen (2 mentions)
Ascorbic acid, by Cyagen (2 mentions)
3 isobutyl 1 methylxanthine, by Cyagen (2 mentions)
Isobutylmethylxanthine, by Cyagen (1 mentions)
Sodium pyruvate, by Cyagen (1 mentions)
Proline, by Cyagen (1 mentions)
L glutamine, by Cyagen (1 mentions)
Tgf β3, by Cyagen (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Alizarin red, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
3 isobutyl 1 methylxanthine ibmx, by Cyagen (1 mentions)
13 protocols using β glycerophosphate
1
Osteogenic Differentiation of Bone Marrow Stem Cells
2
Multilineage Differentiation Assay for Mesenchymal Stem Cells
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For osteogenic differentiation cells were firstly grown to 100% confluence, and then cultured with medium containing α-MEM with 10% FBS, ascorbate, β-glycerophos-phate and dexamethasone (Cyagen Biosciences, Jiangsu, China) for nine days. Medium was changed every 3 days. Alizarin red staining was performed and recorded using an inverted microscope (Olympus, Tokyo, Japan). In addition, mRNA expression of Runx2 and osteocalcin (OCN) was analyzed with qPCR. To assess adipogenic differentiation cells were cultured at 100% confluence for 2∼3 weeks in α-MEM containing 10% FBS, L-glutamine, insulin, isobutylmethylxanthine, rosiglitazone and dexame-thasone (Cyagen Biosciences, Jiangsu, China). Medium was changed every 2∼3 days and then Oil red staining was performed. For chondrogenic differentiation, 3−4×105 suture mesenchyme cells were cultured as a pellet at the bottom of a 15 ml aseptic tube in 0.5 ml α-MEM medium supplemented TGF-β3, dexamethasone, ascorbate, sodium pyruvate and proline (Cyagen Biosciences, Jiangsu, China) and changed every 3 days. After 21 days culture, the cell microsphere was formalin-fixed and paraffin-em-bedded and then sectioned at 4 mm, stained with Alcian blue.
3
Osteogenic Differentiation Potential Assay
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To investigate osteogenic differentiation potential, cells were initially grown under the conditions described above for 48 h. Following this, cells were immediately cultured in commercial osteogenic media supplemented with 50 µg/ml ascorbic acid, 0.1 µM dexamethasone and 10 mM β-glycerophosphate (Cyagen Biosciences, Santa Clara, CA, USA) for 14 days (13 (link),30 (link)). Cells were subsequently fixed in 4% formalin for 30 min at room temperature and stained with alizarin red S (Cyagen Biosciences) for 30 min. To quantify mineralization, the mineral stain was solubilized in 5% SDS in 0.5 ml 0.5 N HCl for 30 min. The absorbance was measured at a wavelength of 405 nm using a microplate reader.
4
Osteogenic and Adipogenic Differentiation of Rat ADSCs
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For osteogenic differentiation, rADSCs at passage 3 were inoculated at 4×103 cells/cm2 and cultured for 2 weeks in osteogenic induction medium [MEM containing 10% FBS, 100 µg/ml ascorbate, 0.1 µM dexamethasone and 10 mM β-glycerophosphate (All from Cyagen Biosciences, Suzhou, Jiangsu, China)]. rADSCs were then fixed with 4% paraformaldehyde (PFA, Boster Biosciences, Beijing, China) for 20 min, washed with PBS for 3 times, afterward, incubated with 1 mg/ml Alizarin Red (Sigma-Aldrich, St. Louis, MO) solution for 30 min to stain for calcium deposition. For adipogenic differentiation, rADSCs at passage 3 were cultured for 2 weeks in adipogenic induction medium [high glucose-DMEM containing 10% FBS, 1 µM dexamethasone, 0.5 mM methyl-isobutylxanthine, 10 µg/ml insulin, and 100 µM indomethacin (All from Cyagen Biosciences, Suzhou, Jiangsu, China)]. Then, cells were fixed with 4% PFA for 20 min, washed with PBS for 3 times, afterward, immersed in 0.3% Oil Red O solution (Sigma-Aldrich, St. Louis, MO) in 60% isopropanol for 30 min to assess lipid droplet formation.
5
Osteogenic and Adipogenic Differentiation of Murine Bone Marrow Mesenchymal Stem Cells
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To identify the role of l ‐THP on osteogenesis and the formation of the calcified nodule, we flushed bilateral femoral bone marrow of 4‐week‐old C57BL/6 mice to isolate bone marrow mesenchymal stem cells (BMSCs). To induce osteogenesis, BMSCs were cultured with complete medium supplied with 100 nmol/L dexamethasone, 50 μmol/L ascorbic acid and 10 mmol/L β‐glycerophosphate (Cyagen Biosciences). Prepared cells were stained with ALP staining (Sigma‐Aldrich) after osteogenic induction for 14 days, while alizarin red staining was conducted after 21 days. To induce adipogenesis, BMSCs were cultured with 10% FBS α‐MEM supplied with 10 μg/mL insulin, 200 μmol/L indomethacin, 1 μmol/L dexamethasone and 0.5 mmol/L 3‐isobutyl‐1‐methylxanthine (IBMX) (Cyagen Biosciences). Differentiated cells were then marked with Oil Red O staining (Sigma‐Aldrich).
6
BMSCs Osteogenic Differentiation
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BMSCs were seeded into 6- or 48-well plates and cultured in alpha-modified Eagle’s medium. After cell attachment, the culture medium was changed to osteogenic medium supplemented with 10% FBS, 50 μg·mL−1 ascorbate, 10 μmol·L−1 β-glycerophosphate, 0.1 μmol·L−1 dexamethasone, and 10 μmol·L−1glutamine (Cyagen Biosciences, Guangzhou, China). The osteogenic induction medium was replaced every 2 days.
7
Osteogenic Differentiation of BMSCs
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The osteogenic differentiation protocol was described in our previous studies [17 (link), 18 (link)]. Briefly, the BMSCs were cultured in growth medium in 24-well plates at a density of 1 × 104 cells/cm2. When over 80% confluence was reached, the medium was replaced with osteogenic induction medium (OIM), which consisted of growth medium supplemented with 1 nM dexamethasone, 50 μM l -ascorbic acid-2-phosphate and 20 mM β-glycerophosphate (Cyagen Biosciences, Guangzhou, China). Catalpol (10, 50 or 250 μM) was added to OIM in the experimental group, while the control group was cultured in OIM without catalpol. In accordance with a previous study [21 (link)], to examine the involvement of WNT/β-catenin signalling, OIM was supplemented with 50 μM catalpol in the presence or absence of 0.1 μg/ml Dickkopf-related protein 1 (DKK1; Peprotech, CT, USA), an antagonist of the WNT/β-catenin pathway.
8
Osteogenic Differentiation of hADSCs
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Passage 3 hADSCs were harvested by trypsin digestion as described above; the cells were counted and seeded at a density of 105 per well in a six-well plate. When the cells reached 100 % confluence, MSC Osteogenic Differentiation Basal Medium containing 10 % FBS, 1 % penicillin-streptomycin, glutamine, ascorbate, β-glycerophosphate, and dexamethasone (Cyagen Biosciences) was added to four wells while complete culture medium was added to the other two wells as the negative controls. The medium was changed every 3 days for 3 weeks. The differentiation potential for osteogenesis was assessed by 40 mM Alizarin Red (pH 4.2) staining.
9
Osteogenic Differentiation of Adipose-Derived Stem Cells
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The cells (passage 3) were transplanted to 6-well cell culture plates. Cells were grown to 50–70% confluence in complete medium after 24–48 h [20 (link)]. The medium was completely replaced with an osteogenic differentiation medium, which consisted of human adipose-derived stem cell osteogenic differentiation basal medium, human adipose-derived stem cell osteogenic differentiation fetal bovine serum, penicillin-streptomycin, glutamine, ascorbate, β-glycerophosphate, and dexamethasone (Cyagen, USA). The cells were then cultured up to 21 days. The osteogenic medium was changed every 72 h.
10
Adipogenic and Osteogenic Differentiation of MSCs
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First, MSCs were grown in complete medium for adipogenic differentiation. When the cells reached 80% confluence, the medium was changed to adipogenic medium that consisted of rosiglitazone, 3-isobutyl-1-methylxanthin (IBMX), glutamine, dexamethasone and insulin (Cyagen Biosciences, Suzhou, China). Osteogenic differentiation was induced in MSCs by DMEM containing dexamethasone, ascorbic acids and β-glycerophosphate (Cyagen Biosciences, Suzhou, China). For Oil Red O staining, MSCs were fixed with 4% formaldehyde. After being washed with PBS, the cells were incubated with Oil Red O solution (Cyagen Biosciences Suzhou, China) for 30 min. The cells were then washed and examined under a microscope. For Alizarin Red S staining, the cells were incubated with Alizarin Red Solution (Cyagen Biosciences, Suzhou, China) for 30 min. Images were acquired by a microscope (Supplementary Figure S1B ).
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