To study the performance of SPIO-Au NPs in osteogenic differentiation of MC3T3-E1 cells, the ALP activity level was assessed by using an Abcam® Alkaline phosphatase assay kit (ab83369, Abcam, Cambridge, MA). MC3T3-E1 cells were seeded into a 24-well plate at a density of 5*104 cells per well with growth medium. After 24 h of incubation, the medium was changed to osteogenic induction medium, containing SPIO-Au NPs at different concentrations (0, 1, 5, 10 and 20 μg/mL). The osteogenic induction medium consisted of αMEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), 1% penicillin (Gibco, Grand Island, NY), 10 mM disodium β–glycerophosphate (Sigma-Aldrich, St. Louis, MO), 10 nM dexamethasone (Sigma-Aldrich, St. Louis, MO) and 0.28 mM ascorbic acid (Sigma-Aldrich, St. Louis, MO). All the cells were incubated for 7, 10 and 14 days. At each time period, cells were harvested following the manufacturer’s instruction. The intensity was measured at the wavelength of 405 nm on a microplate reader (SpectraMax i3x, Molecular Devices Inc.). The amount of ALP in each well was calculated and normalized by total protein content, which was measured simultaneously using the Pierce™ Coomassie (Bradford) protein assay kit (Thermo Scientific, Rockford, IL).
β glycerophosphate disodium
Manufactured by Merck Group
Sourced in United States
β-glycerophosphate disodium is a chemical compound commonly used as a buffer in various laboratory applications. It is a white, crystalline powder that is soluble in water. The primary function of β-glycerophosphate disodium is to maintain a stable pH environment in laboratory experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (7 mentions)
Ascorbic acid, by Merck Group (6 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Pierce coomassie bradford protein assay kit, by Thermo Fisher Scientific (1 mentions)
Spectramax i3x, by Molecular Devices (1 mentions)
Rifampicin, by Thermo Fisher Scientific (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Yeast extract, by Merck Group (1 mentions)
Brain heart infusion media, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Galactose, by Merck Group (1 mentions)
Todd hewitt broth, by Merck Group (1 mentions)
Dfc3000 g microscope, by Leica (1 mentions)
Recombinant human bmp 2, by R&D Systems (1 mentions)
α mem, by Fujifilm (1 mentions)
Ascorbic acid 2 phosphate, by Merck Group (1 mentions)
Alp activity kit, by Nanjing Jiancheng (1 mentions)
Hydroxypropyl cellulose, by Merck Group (1 mentions)
14 protocols using β glycerophosphate disodium
1
Evaluating SPIO-Au NPs' Osteogenic Potential
2
Genotyping and culture conditions for S. pneumoniae
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Genotypes used in this study are described in Supplementary Table S1 . Unless otherwise stated, encapsulated S. pneumoniae were cultured statically at 35°C in 5% CO2. Culturing on solid media used Todd-Hewitt broth supplemented with 0.5% yeast extract and 1.5% agar (Sigma-Aldrich). Media were supplemented antibiotics for selection of mutated genotypes: rifampicin (Fisher Scientific) at 4 μg ml−1; kanamycin (Sigma-Aldrich) at 400 μg ml−1, or chloramphenicol (Sigma-Aldrich) at 4 μg ml−1. Phase contrast microscopy of colonies used a Leica DFC3000 G microscope.
Unless otherwise specified, culturing in liquid media used 10 ml of a 2:3 ratio mixture of Todd-Hewitt broth (Sigma-Aldrich) with 0.5% yeast extract (Sigma-Aldrich), and Brain-Heart Infusion media (Sigma-Aldrich); this is referred to as ‘mixed’ media. Transformation experiments with S. pneumoniae R6 derivatives used a chemically-defined medium, consisting of disodium β-glycerophosphate (20 g l−1; Sigma-Aldrich), sodium pyruvate (0.1 g l−1; Fluorochem), choline (0.001 g l−1; Alfa Aesar), cysteine (0.4 g l−1; Tokyo Chemical Industry UK), glucose (3.8 mM; Sigma-Aldrich) and galactose (12 mM; Sigma-Aldrich). Carbon source supplements were added to liquid media at a final concentration of 30 mM, unless otherwise specified.
Unless otherwise specified, culturing in liquid media used 10 ml of a 2:3 ratio mixture of Todd-Hewitt broth (Sigma-Aldrich) with 0.5% yeast extract (Sigma-Aldrich), and Brain-Heart Infusion media (Sigma-Aldrich); this is referred to as ‘mixed’ media. Transformation experiments with S. pneumoniae R6 derivatives used a chemically-defined medium, consisting of disodium β-glycerophosphate (20 g l−1; Sigma-Aldrich), sodium pyruvate (0.1 g l−1; Fluorochem), choline (0.001 g l−1; Alfa Aesar), cysteine (0.4 g l−1; Tokyo Chemical Industry UK), glucose (3.8 mM; Sigma-Aldrich) and galactose (12 mM; Sigma-Aldrich). Carbon source supplements were added to liquid media at a final concentration of 30 mM, unless otherwise specified.
3
Osteogenic Differentiation of Pericytes
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Pericytes were seeded in 24-well plates at 1 × 104 cells/well. Cells were cultured in Minimum Essential Medium Eagle (α-MEM, Wako) containing 10% FBS, 50 μM ascorbic acid 2-phosphate (Sigma-Aldrich), 5 mM disodium β-glycerophosphate (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich) and 0.3 mg/mL recombinant human BMP-2 (R&D Systems, Minneapolis, MN, USA) for 6–9 days. The culture medium was changed every 3 days, and ALP activity and mineralization potential were evaluated by an ALP assay and von Kossa staining, respectively, as previously described [38 (link)].
4
Osteogenic Differentiation of MC3T3-E1 Cells
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MC3T3-E1 cells were seeded in 24-well plates (2×104 cells/well) containing α-MEM medium and 10% FBS. After 24 h, the culture medium was changed to α-MEM, 10% FBS and osteogenic induction supplement containing 10 mmol/l disodium β-glycerophosphate and 0.15 mmol/l ascorbic acid (Sigma, St. Louis, MO, USA). A series of dilutions of BD (final concentrations, 1×10−6–1×10−9 M) were added to the culture medium in the 24-well plates for 3, 5, 7, 10 and 14 days. MC3T3-E1 cells treated with only osteogenic induction supplement were used as the control group. Following incubation, the MC3T3-E1 cells were washed twice with ice-cold PBS and lysed by two cycles of freezing and thawing. Aliquots of the supernatants were subjected to ALP activity and protein content measurement using an ALP activity kit and a bicinchonininc acid (BCA) protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). All the results were normalized by protein content.
5
Chitosan-based Biomaterial Formulations
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The studies used chitosan of crab (Sigma Aldrich, Poznan, Poland, product No. 50494) and shrimp (Sigma Aldrich, Poznan, Poland, product No. 50494) origin with different molecular weight, hydroxypropyl cellulose (Sigma Aldrich, Poznan, Poland, product No. 191884), hydrochloric acid (Fluka Analitycal provided by Alchem, Torun, Poland, product number: 84415), and disodium β-glycerophosphate (Sigma Aldrich, Poznan, Poland, product No. 50020).
6
Cell Differentiation and Stimulation Assay Protocols
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Cell culture reagents, including fetal bovine serum (FBS; 10099141C), minimum essential medium α (α-MEM; C12571500BT), and penicillin/streptomycin (P/S; 15140122), were all purchased from Gibco (USA). Reagents for cell differentiation and stimulation included lipopolysaccharide (L8274; Sigma-Aldrich), cyclic polypeptide D7 (Scilight-Peptide Inc.), ascorbic acid (A8960; Sigma-Aldrich), β-glycerophosphate disodium (G9422; Sigma-Aldrich), dexamethasone (HY-14648; MCE), macrophage colony-stimulating factor (M-CSF; 416-ML-050; R&D Systems), and receptor activator of nuclear factor-κB ligand (RANKL; 462-TEC-010; R&D Systems). Primary antibodies that used in western blotting (WB) for the detection of ALP (ab229126), Runx2 (ab236639), and Ctsk (ab187647) were from Abcam; for Col1α1 (72026t) detection was from Cell Signaling Technology; for Nfatc1 (sc-7294) and Traf6 (sc-8409) detections were from Santa Cruz; for β-actin (hrp-66009) detection was from Proteintech. Secondary antibodies were purchased from Proteintech, including anti-rabbit (SA00001-2) and anti-mouse (SA00001-1).
7
Osteogenic Differentiation of Mesenchymal Stem Cells
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Human bone marrow mesenchyme stem cells were seeded at 12‐well culture plates and grew to confluence with MSCM medium. Then, the MSCM medium was removed, and osteogenic induction medium composed of MEM containing 10% FBS, 10 mmol/L β‐glycerophosphate disodium (Sigma, G9422), 0.2 mmol/L ascorbic acid (Sigma, A5960), 0.1 µmol/L dexamethasone (Sigma, D1756)26 was added to induce osteogenic differentiation. CHS and Si ion were added in osteogenic induction medium, and HBMSCs were cultured in these media for 7 days. Then, alkaline phosphatase (ALP) kit purchased from Beyotime Biotechnology, Shanghai (P0321), was used to stain the cells to evaluate the ALP protein synthesis in the HBMSCs cultured with different media.
8
Osteogenic Differentiation of BM-hMSCs
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BM-hMSCs were isolated as previously described17 (link). For this study we have used mainly cells at passage 3. For proliferation, cells were cultured at 37 °C in a humidified incubator with 5% CO2 in maintenance medium (MM), low-glucose DMEM (Dulbecco’s modified Eagle’s) supplemented with 10% FBS, 1% glutamine, 50 μg/ml penicillin-streptomycin and amphotericin B (Lonza Group Ltd.). To induce osteogenesis, BM-hMSCs were maintained in Osteogenic Differentiating Medium (ODM), α-MEM (Minimum Essential Medium) supplemented with 10% FBS, antibiotics and the osteogenic mixture containing 100nM dexamethasone, 5 mM β-glycerophosphate disodium and 50 mg/ml ascorbic acid (Sigma-Aldrich, S. Louis, MO, USA). Treatment lasted up to 27 days and the medium was changed every 3 days. The study was conducted in accordance with the Review Board of Fondazione IRCCS Policlinico San Matteo and the University of Pavia (2011).
9
Osteogenic Differentiation of Adipose-derived MSCs
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Adipose-derived MSCs were obtained from Lonza (Walkersville, MD, USA) and cultured in serum free StemPro® MSC SFM XenoFree medium (Thermo Fisher Scientific, Waltham, MA, USA) containing CTS™ GlutaMAX™-I (Thermo Fisher Scientific) at 37 °C in an atmosphere with 5% CO2. For osteogenic differentiation, passage 3–6 ADSCs were cultured to 90% confluence, and then the culture medium was replaced with osteogenic medium [MSC growth medium supplemented with 5 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 250 μM (+)-sodium L-ascorbate (Sigma-Aldrich), and 10 mM β-glycerophosphate disodium (Sigma-Aldrich)]. The medium was changed two or three times a week, and the cells were cultured for 21 days.
10
Osteogenic Differentiation of MSCs
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MSCs were seeded in 24-well plates at a density of 1 × 104 cells/well in growth medium. After 24 h, the medium was changed to OIM that comprised DMEM (Gibco, USA) supplemented with 10% FBS, 10−7 M dexamethasone (Sigma, USA), 10 mM β-glycerophosphate disodium (Sigma, USA), and 50 μg/ml L-Vc (Sigma). The medium was replaced every 3 days. After induction, mineralized nodules were detected by alizarin red S staining (ARS, Sigma). Alkaline phosphatase (ALP) enzymatic activity was analyzed using an NBT/BCIP alkaline phosphatase color development kit (Beyotime, China).
For coculture of MSCs and HUVECs, MSCs were seeded in 24-well plates at a density of 1 × 104 cells/well, while HUVECs were seeded at a density of 2 × 104 cells/well. The initial medium was a 1:1 (v/v) mixture of α-MEM and ECM supplemented with 10% FBS and 100× ECGS. After 24 h, the medium was changed to OIM supplemented with 100× ECGS.
For coculture of MSCs and HUVECs, MSCs were seeded in 24-well plates at a density of 1 × 104 cells/well, while HUVECs were seeded at a density of 2 × 104 cells/well. The initial medium was a 1:1 (v/v) mixture of α-MEM and ECM supplemented with 10% FBS and 100× ECGS. After 24 h, the medium was changed to OIM supplemented with 100× ECGS.
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