The following analytical-grade chemicals were purchased from commercial sources and used without further purification: bicarbonate buffer (BB; 0.1M, pH 8.4), ferric chloride hexahydrate, hydrochloric acid (1 M), sodium hydroxide (1 M), sodium nitrate, Triton X-100, gelatin from porcine skin, human serum albumin (HSA), NHS-Cy7, rhodamine isothiocyanate (RITC), divinyl sulfone (DVS), triethylamine (TEA), D-glucose from Sigma (Israel); FGF2 ELISA kit and recombinant human FGF2 from PeproTech (Israel); Midi-MACS magnetic columns from Almog Diagnostic (Israel); phosphate-buffered saline (PBS free of Ca+2 and Mg+2; 0.1 M, pH 7.4) from Biological-Industries (Israel); tissue culture plates (96 wells) and plastic tips from Greiner bio-one (Germany); Water was purified by passing deionized water through an Elgastat Spectrum reverse osmosis system (Elga, High Wycombe, UK). All tissue culture reagents were from Biological Industries (Israel). B27 and DAPI were from Invitrogen. Dexamethasone, insulin, β-glycophostphate, ascorbate phosphate, neuron-specific microtubule-associated protein 2 mouse monoclonal antibody and dyes were from Sigma. Glial Fibrillary Acidic Protein rabbit monoclonal antibody was from Cell Signaling. TUNEL TMR Red was from Roche. Secondary antibodies were from Jackson ImmunoResearch. PCNA (pc1-0) mouse monoclonal IgG2a was from Santa Cruz, USA.
Ascorbate phosphate
Manufactured by Merck Group
Sourced in United States, Germany
Ascorbate phosphate is a chemical compound used as a lab equipment product. It functions as a source of ascorbic acid, which is a essential nutrient and antioxidant. The core function of ascorbate phosphate is to provide a stable and water-soluble form of vitamin C for various research and experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (13 mentions)
β glycerophosphate, by Merck Group (7 mentions)
β glycerol phosphate, by Merck Group (4 mentions)
Insulin, by Merck Group (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Indomethacin, by Merck Group (2 mentions)
Isobutylmethylxanthine, by Merck Group (2 mentions)
Rhodamine isothiocyanate, by Merck Group (1 mentions)
Divinyl sulfone, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Sartorius (1 mentions)
Tunel tmr red, by Roche (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by GE Healthcare (1 mentions)
14 protocols using ascorbate phosphate
1
Comprehensive Chemical Reagents for Cell Culture
2
Osteogenic Differentiation of Human MSCs
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In the present study, human MSCs (Lonza Biologics plc, Cambridge, UK) were maintained in a standard medium consisting of the α-minimum essential medium (α-MEM) (Gibco Life Technologies Ltd, Paisley, UK) containing 15% fetal calf serum (FCS) (PAA Laboratories, Yeovil, UK) supplemented with 200 U ml−1 penicillin, 200 µg ml−1 streptomycin and 2 mM L-glutamine (all from Gibco) at 37°C in a humidified atmosphere of 5% CO2 in air. The media were changed every 2–3 days. Cells between passages 4–6 were used in the study.
For osteogenic differentiation induction, MSCs were seeded at a density of 1.5 × 104 cells cm−2 and allowed to grow with the standard medium for the first 48 h until the cells reached 80% confluence. Then, the cells were incubated with an osteogenic medium (OM) (standard medium with 100 nM dexamethasone, 50 µM ascorbate-phosphate and 10 mM β-glycerolphosphate) (all from Sigma-Aldrich, St Louis, MO, USA).
For osteogenic differentiation induction, MSCs were seeded at a density of 1.5 × 104 cells cm−2 and allowed to grow with the standard medium for the first 48 h until the cells reached 80% confluence. Then, the cells were incubated with an osteogenic medium (OM) (standard medium with 100 nM dexamethasone, 50 µM ascorbate-phosphate and 10 mM β-glycerolphosphate) (all from Sigma-Aldrich, St Louis, MO, USA).
3
Characterization of hJBMMSCs Differentiation
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Before seeded on scaffolds, hJBMMSCs were examined for their capacity to differentiate into either adipocytes or osteocytes. Osteogenesis differentiation was induced by incubating cells at a density of 4 × 103 cells/cm2 in osteogenic medium containing 10 mM β-glycerophosphate, 10 nM dexamethasone, 50 mg/mL ascorbate phosphate (all from Sigma-Aldrich, MO, USA), and 10% FBS for 4 weeks. Mineral deposits indicating osteogenesis differentiation were stained with 40 mM Alizarin Red S (Sigma-Aldrich, MO, USA). Adipogenesis differentiation was induced by incubating cells at a density of 4 × 103 cells/cm2 in adipogenic medium containing 1 mM dexamethasone, 0.2 mM indomethacin, 0.01 mg/mL insulin and 0.5 mM isobutyl-methylxanthine (all from Sigma-Aldrich, MO, USA), and 10% FBS for 5 weeks. Lipid vacuoles indicating adipogenesis differentiation were stained with Oil Red staining (Sigma-Aldrich, MO, USA).
4
Odontoblastic Differentiation Induction Protocol
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For odontoblastic differentiation, the O9-1 cells and iNCLCs were cultured on collagen I (Sigma)-coated dishes. Cells were incubated with odontogenic induction medium-DMEM (Invitrogen) supplemented with 100 nM dexamethasone (Sigma), 5 mM β-glycerophosphate (Sigma-Aldrich), 50 μg/ml ascorbate phosphate (Sigma-Aldrich), 10% FBS (Invitrogen), and 100 ng/ml BMP-4 (R&D). The expression of odontogenic-related gene including Msx-1, Runx-2, Dmp-1, and Dspp were evaluated using real-time RT-PCR on day 10. The corresponding primer sequences are displayed in Table 1 . Alkaline phosphatase (ALP) staining was performed using an ALP kit (Beyotime, China) on day 10 (Zhang M. et al., 2018 (link)).
5
Osteogenic Differentiation Assay for U2OS and 143B Cells
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U2OS and 143B cells were induced for osteogenic differentiation in a normal medium supplemented with 10 mM β-glycerophosphate (Sigma, St. Louis, USA), 50 mg/ml ascorbate phosphate (Sigma), and 10 nM dexamethasone (Sigma). After 5 days of induction, cells were fixed with 4% PFA, washed with PBS, and stained using BCIP/NBT ALP Color Development Kit (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer’s protocols. After 14 days, Alizarin red (Beyotime Institute of Biotechnology, Shanghai, China) was used to stain cells. Photos were taken using a scanner and light microscope (DMIL LED; Leica Microsystems GmbH) at ×200 magnification.
6
Differentiation of MSCs to Adipocytes and Osteocytes
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We verified the differentiation potential of MSCs to osteocytes and adipocytes. Differentiation to adipocytes was induced by culturing MSCs in DMEM supplemented with 10% FBS, 1 mM dexamethasone (Sigma, USA), 200 mM indomethacin (Sigma, USA), 1.7 mM insulin (Sigma, USA), 500 mM isobutyl-methylxanthine (Merck, Germany), 0.05 U/ml penicillin, and 0.05 mg/ml streptomycin for 2 weeks. We have used oil red-O staining (Sigma, USA) to identify the presence of adipocytes. oil red-O staining visualizes intracellular lipid accumulation. For staining, the cells were fixed in 10% formaldehyde (Merck, Germany) for 1 hour, after which they were washed with 60% isopropanol, (Merck, Germany) and stained with oil red-O solution in 60% isopropanol for 10 minutes. Next, cells were washed with distilled water and de-stained in 100% isopropanol for 15 minutes.
Differentiation of MSCs to osteocytes was induced Stem Cell Derived Endothelial Cells for Neovascularization in α-MEM (Gibco, USA) that contained 10% FBS, 0.1 mM dexamethasone, 10 mM β-glycerophosphate (Sigma, USA), and 50 mM ascorbate-phosphate (Sigma, USA) for two weeks. A specific histochemical stain for alkaline phosphatase (ALP) with an alkaline phosphatase staining kit (Sigma Chemical Co., USA) was used to identify the osteocytes.
Differentiation of MSCs to osteocytes was induced Stem Cell Derived Endothelial Cells for Neovascularization in α-MEM (Gibco, USA) that contained 10% FBS, 0.1 mM dexamethasone, 10 mM β-glycerophosphate (Sigma, USA), and 50 mM ascorbate-phosphate (Sigma, USA) for two weeks. A specific histochemical stain for alkaline phosphatase (ALP) with an alkaline phosphatase staining kit (Sigma Chemical Co., USA) was used to identify the osteocytes.
7
Osteogenesis Induction and Dexamethasone Response
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Bone marrow was flushed from hind legs of C57BL/6 or GR mutant mice and seeded at a density of 1,000,000 cells per cm² in DMEM containing 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (Pen/Strep) (all from Invitrogen, Karlsruhe, Germany). At 70% confluence, cells were switched to differentiation media with 5 mM β-glycerol phosphate and 100 µM ascorbate phosphate (all from Sigma-Aldrich) for 7 days. Thereafter, cells were switched to starving media (DMEM containing 1% FCS and 1% Pen/Strep) overnight and treated with DEX (Sigma-Aldrich) at concentrations as indicated in the figure legends for a maximum of 48 hours. In some experiments, cells were co-treated with 1 µM RU-486 (Sigma-Aldrich), an established GR antagonist.
8
Osteogenic and Adipogenic Differentiation of MSCs
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RGN-MSCs and RGI-MSCs were seeded into 6-well plates at 1×105 cells/well and cultured in 10% FBS-containing DMEM with either osteogenic (0.1 µM dexamethasone, 10 µM β-glycerophosphate, 50 µM ascorbate-phosphate; Sigma, St. Louis, MO, USA) or adipogenic supplements (Cyagen, Guangzhou, China). The medium was changed every 3 days and the cells were induced for 2 weeks. At the end of induction, the cells were subjected to neutrophil alkaline phosphatase (NAP) staining or Oil Red O staining.
9
In Vitro Osteogenic Differentiation Assay
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For osteogenic differentiation, the cells (P4) were seeded in 6-well plates at a density of 4×104 cells/per well, which aimed to quantify mineralized nodule formation in vitro. The cells were incubated in medium supplemented with 10% FBS. The medium was then switched to osteogenic differentiation medium, which contains 0.1 mM dexamethasone, 10 mM β-glycerophosphate, 50 mg/mL ascorbate phosphate (all from Sigma Aldrich), and 10% FBS. The medium was changed every 2 days. Mineralization was detected and quantified using an Alizarin Red, and 4 wells were analyzed for each group. The quantification assay was performed using an ALP activity detection kit (Jiancheng Bioengineering, Nanjing, China). The cells were fixed with 4% paraformaldehyde for 20 min and stained with 2% Alizarin Red (PH 4.2) (Kermel, Tianjin, China). The mineralized nodules were dissolved by hexadecyl pyridinium chloride and isopropanol, and quantitative absorbance was measured at 560nm for statistical analysis.
10
Osteogenic Differentiation of Murine and Human Cells
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Over a time period of 18 days, pre-osteoblastic, murine MC3T3-E1 cells and hMSC were cultured in osteogenic medium (α-MEM, Biochrom, Germany) containing 10% fetal calf serum (Biochrom AG), 1% penicillin/streptomycin (PAA, Germany), 2 mM L-glutamine (PAA), 100 μM dexamethasone (Sigma-Aldrich, Germany), 10 mM β-glycerol phosphate (Sigma-Aldrich), 100 μM ascorbate phosphate (Sigma-Aldrich), and 5 ng/ml BMP-2 (PeproTech, Germany) at 37 °C and 5% carbon dioxide [31 (link)]. Therefore, 8 μl of cell suspension, consisting of the cell culture medium and 100.000 cells, was applied on each scaffold and incubated for 20 min to allow cell attachment to scaffold surface. Afterwards, wells were filled up with cell culture medium until the scaffolds were completely covered. Every other day, the scaffold was transferred into a new well and the cell culture medium was changed. Culturing hMSC was approved by the ethics committee of the Faculty of Medicine of the Technische Universität Dresden (EK 245082010).
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