The osteogenetic differentiation medium was StemPro Osteogenesis Differentiation Kit from Invitrogen. The level of ALP activity was determined after the cells had been seeded for 3 and 7 days. The process was as follows: the cells were lysed from discs using 0.2% NP-40 and centrifuged for 10 min at 2000 rpm after washing with PBS. ALP activity was determined using p-nitrophenyl phosphate (pNPP, Sigma) as the substrate. Each sample was mixed with pNPP in 1 M diethanolamine buffer for 15 min, after which the reaction was stopped by the addition of 5 N NaOH and quantified by absorbance at 405 nm. All experiments were done in triplicate. The osteogenic differentiation medium was Advance MEM (Thermo Fisher Scientific Inc.) supplemented with 10−8 M dexamethasone (Sigma-Aldrich), 0.05 g/L l -Ascorbic acid (Sigma-Aldrich) and 2.16 g/L glycerol 2-phosphate disodium salt hydrate (Sigma-Aldrich). The OC protein released from the hMSCs was cultured on different substrates for 7 and 14 days after cell seeding. Following the manufacturer’s instructions, an osteocalcin enzyme-linked immunosorbent assay kit (Invitrogen) was used to determine the OC protein content. The OC protein concentration was measured by correlation with a standard curve. The analyzed blank disks were treated as controls. All experiments were done in triplicate.
Glycerol 2 phosphate disodium salt hydrate
Manufactured by Merck Group
Sourced in United States
Glycerol 2-phosphate disodium salt hydrate is a chemical compound used in various laboratory applications. It serves as a source of glycerol and phosphate ions. The product is available in a crystalline hydrated form.
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Lab products found in correlation
Dexamethasone (dex), by Merck Group (4 mentions)
L ascorbic acid, by Merck Group (3 mentions)
Stempro osteogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
Amyloglucosidase, by Merck Group (1 mentions)
Glucose 6 phosphate (g6p), by Merck Group (1 mentions)
Ascorbic acid, by Fujifilm (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Heat inactivated fetal calf serum, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Tgf β3, by Novoprotein (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
L proline, by Thermo Fisher Scientific (1 mentions)
Oc enzyme linked immunosorbent assay kit, by Thermo Fisher Scientific (1 mentions)
P nitrophenyl phosphate, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Mem alpha medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Polyvinyl acetate pvac, by Merck Group (1 mentions)
7 protocols using glycerol 2 phosphate disodium salt hydrate
1
Osteogenic Differentiation of hMSCs
2
Quantitative and Qualitative Analysis of Hepatic G6Pase Activity
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G6Pase enzyme analysis was performed as previously described on liver tissues collected at necropsy.21 (link) Tissues were frozen and stored at −80°C. G6Pase activity was quantified by using glucose-6-phosphate (Sigma, St. Louis, MO, USA) as substrate after subtraction of nonspecific phosphatase activity as estimated by glycerol 2-phosphate disodium salt hydrate (Sigma, St. Louis, MO, USA). G6Pase was assessed qualitatively in flash-frozen sections of dog liver by an optimized cerium-diaminobenzidine method as described.16 (link) Glycogen content was measured by complete digestion of polysaccharide using amyloglucosidase (Sigma, St. Louis, MO, USA). The structure of the polysaccharide was inferred by using phosphorylase free of the debranching enzyme to measure the yield of glucose-1-phosphate.
3
Osteogenic Differentiation Mediated by Lansoprazole
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The growth medium consisted of Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO) supplemented with 100 unit/mL penicillin, 100 µg/mL streptomycin, 0.75 μg/mL fungizone (Gibco, Grand Island, NY), and 10% heat-inactivated fetal calf serum (Thermo Fisher Scientific, Waltham, MA). The osteogenic differentiation medium consisted of the growth medium supplemented with 10−7 M Dexamethasone (Sigma-Aldrich), 10 mM Glycerol-2-phosphate disodium salt hydrate (Sigma-Aldrich), 200 μM Ascorbic acid (FUJIFILM Wako Pure Chemical Corp., Ltd., Osaka, Japan). A water-soluble form of lansoprazole was purchased from Takeda Pharmaceutical Company Limited (Osaka, Japan). Since we previously identified that 20 µM of lansoprazole exerted the maximum effect of upregulating Runx2 and terminal osteoblastic differentiation without apparent cytotoxicity in vitro, we adopted a final concentration of 20 µM as a default one of soluble lansoprazole in this study.
4
Differentiation of Mesenchymal Stem Cells
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For adipogenic differentiation, the confluent cells were cultured with adipogenic medium containing α-DMEM with 10% FBS supplemented with 10 μg/ml insulin, 100 μM indomethacin, 0.5 mM 3-iso- butyl-1-methylxanthine, and 0.1 μM dexamethasone (Sigma, St. Louis, MO). The medium was changed every 2 days during 2 weeks. For osteogenic differentiation, the cells were plated at confluence in osteogenic medium containing α-DMEM with 10% FBS supplemented with 0.1 μM dexamethasone, 0.2 mM L-ascorbic acid, and 10 mM glycerol 2-phosphate disodium salt hydrate (Sigma, St. Louis, MO). The medium was changed every 2 days during 1 or 2 weeks. For chondrogenic differentiation, the cells were resuspended at a concentration of 5 × 105 cells per mL in chondrogenic medium consisting of high-glucose DMEM, 1% ITS(Gibco), 100 nM dexamethasone (DEX, Sigma), 1% P/S, 10 ng/ml TGF-β3 (Novoprotein), 50 μg/ml L-ascorbic acid 2-phosphate (ascorbate), 40 μg/ml L-Proline (proline), and 100 μg/ml sodium pyruvate (Gibco). Cells were then centrifuged for 5 min at 300 ×g to form a pellet. The medium was changed every 2 days during 3 weeks. Chondrogenic pellets were cultured at 37 °C for up to the timepoints required for various experiments.
5
ALP Activity and Osteocalcin Release Assay
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The level of alkaline phosphatase (ALP) activity was determined after the cells had been seeded for 3 days and 7 days. The process was as follows: the cells were lysed from discs using 0.2% NP-40 and centrifuged for 10 minutes at 2000 rpm after washing with PBS. ALP activity was determined using p-nitrophenyl phosphate (Sigma-Aldrich) as the substrate. Each sample was mixed with p-nitrophenyl phosphate in 1M diethanolamine buffer for 15 minutes, after which the reaction was stopped by the addition of 5N NaOH and quantified by absorbance at 405 nm. All experiments were done in triplicate. The osteogenic differentiation medium was Advance Minimum Essential medium (Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10−8M dexamethasone (Sigma-Aldrich), 0.05 g/L L-Ascorbic acid (Sigma-Aldrich), and 2.16 g/L glycerol 2-phosphate disodium salt hydrate (Sigma-Aldrich).
The osteocalcin (OC) protein released from the pulp cells was cultured on different substrates for 7 days and 14 days after cell seeding. Following the manufacturer's instructions, an OC enzyme-linked immunosorbent assay kit (Invitrogen) was used to determine the OC protein content. The OC protein concentration was measured by correlation with a standard curve. The analyzed blank disks were treated as controls. All experiments were done in triplicate.
The osteocalcin (OC) protein released from the pulp cells was cultured on different substrates for 7 days and 14 days after cell seeding. Following the manufacturer's instructions, an OC enzyme-linked immunosorbent assay kit (Invitrogen) was used to determine the OC protein content. The OC protein concentration was measured by correlation with a standard curve. The analyzed blank disks were treated as controls. All experiments were done in triplicate.
6
Osteoblast Differentiation of MC3T3-E1 Cells
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The MC3T3-E1 cell line, subclone 4 [39, 40] established from normal newborn mouse calvaria, was obtained from ATCC (LGC Standards S.r.l., Sesto San Giovanni, Milan, Italy). Cells were cultured in alpha-MEM medium (Invitrogen, Milan, Italy), supplemented with 10% FBS (Sigma-Aldrich, Milan, Italy) and 1% streptomycin/penicillin (Sigma-Aldrich) in 75-cm 2 flasks at a density of 400 000 cells/cm2. When cells reached 80% confluence, 50 mg/ml ascorbic acid and 3 mM glycerol 2-phosphate disodium salt hydrate (Sigma-Aldrich) were added to the medium. Medium was changed every other day. After 5 days of incubation with ascorbic acid-glycerol phosphate (AA/GP), the cells were trypsinized and replated at a density of 7000 cells/cm 2 and cultured with basal medium plus AA/GP and 10 μM retinoic acid (All Trans Retinoic Acid, ATRA, from Sigma-Aldrich) to obtain Os [24, 25, 41] . Alternatively cells were cultured with basal medium plus AA/GP to obtain Ob. The cell populations were then studied at several time points, namely after 4, 8, 12 days of treatment.
7
Biomaterial-based 3D Cell Culture Conditions
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Dulbecco Modified Eagle Medium, alpha-MEM, penicillin/ streptomycin, DPBS were obtained from Lonza. Ascorbic acid 2-phosphate and glycerol 2-phosphate disodium salt hydrate were purchased from Sigma. Fetal bovine serum and M199 medium was obtained from Gibco. ECGS/H were from Promocell. VEGF, TGFb 3 were from Peprotech. Poly(lactide-co-glycolide) (PLGA) (50 : 50 Resomer RG 502, RG 505, RG 504, and 85 : 15 PLGA ester terminated M w 50 000-75 000) and tween 20, biotin, chitosan (CS) (from crab shells), polyethylenimine (PEI) (average M w = 800 Da), poly(L-lactic acid) (PLLA) (viscosity B1.0 dL g À1 ) and poly(vinyl acetate) (PVAc) (average M w = 140 kDa), palmitic acid N-hydroxysuccinimide ester (NHS-palmitic acid) and avidin were from Sigma. All other reagents were from Sigma and used without further purification. Analytical grade chloroform, glacial acetic acid, dichloromethane (DCM) were from Thermo Fisher Scientific, UK. Emulsions were generated using a Branson sonifier 250 at 40% max amplification. 1 H nuclear magnetic resonance spectra were recorded on a Bruker AVA500 spectrometer (500 MHz respectively) at 298 K in deuterated solvents.
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