The following materials were used: Rosuvastatin (sipla, india), cholestrol (merck, Germany), span20 (merck, Germany), span40 (merck,Germany), span60 (merc,Germany), Sodium hyaluronate (contipro, Czech Republic), Chloroform, Ethanol and all other chemicals and solvents (merck,Germany). Dental pulp stem cells (Bonyakhte,Iran), β-glycerolphosphate (Merck,Germany), ascorbic acid biphosphate (Sigma‐Aldrich,USA) and dexamethasone (Sigma‐Aldrich, USA), Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), penicillin/streptomycin, β‐glycerolphosphate (Merck), ascorbic acid biphosphate (Sigma‐Aldrich), dexamethasone (Sigma‐Aldrich).
β glycerol phosphate
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, Italy, China, Sao Tome and Principe, Australia, Switzerland, Spain
β-glycerol phosphate is a compound commonly used as a buffer in laboratory applications. It helps maintain a stable pH environment in various biochemical and cell culture experiments.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (277 mentions)
Ascorbic acid, by Merck Group (184 mentions)
L ascorbic acid, by Merck Group (57 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (49 mentions)
Indomethacin, by Merck Group (48 mentions)
Alizarin red s, by Merck Group (39 mentions)
Insulin, by Merck Group (36 mentions)
Ascorbate 2 phosphate, by Merck Group (31 mentions)
Oil red o, by Merck Group (29 mentions)
Streptomycin, by Thermo Fisher Scientific (29 mentions)
Penicillin, by Thermo Fisher Scientific (29 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (28 mentions)
α mem, by Thermo Fisher Scientific (28 mentions)
Ascorbic acid 2 phosphate, by Merck Group (26 mentions)
Alizarin red, by Merck Group (22 mentions)
Isobutylmethylxanthine, by Merck Group (21 mentions)
L ascorbic acid 2 phosphate, by Merck Group (20 mentions)
Fetal bovine serum (fbs), by Merck Group (19 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (17 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (15 mentions)
419 protocols using β glycerol phosphate
1
Dental Pulp Stem Cell Osteogenesis
2
Mesenchymal Stem Cell Differentiation
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The osteogenic and adipogenic differentiation capacity of AL-MSCs and HD-MSCs was evaluated at passage P2/P3 [24 (link)]. For osteogenic differentiation, the induction medium was αMEM, 10% FBS, 10−7 M dexamethasone, 50 mg/mL L-ascorbic acid, and 5 mM β-glycerol phosphate (all from Sigma Aldrich, St. Louis, MO, USA); for adipogenic differentiation, the induction medium was αMEM, 10% FBS, 10−7 M dexamethasone, 50 mg/mL L-ascorbic acid and 5 mM β-glycerol phosphate, 100 mg/mL insulin, 50 mM isobutyl methylxanthine (Sigma-Aldrich), and 0.5 mM indomethacin (MP Biomedica, Illkirch, France). In both protocols, differentiation was evaluated after 21 days. In vitro osteogenic differentiation was evidenced by phosphatase alkaline activity stained in blue/violet by BCIP/NBT and calcium deposition stained by Alizarin Red S (both from Sigma-Aldrich). In vitro adipogenic differentiation was evidenced by the appearance of fat droplets stained with Oil Red O (Bio Optica, Milan, Italy).
3
MSC Differentiation Potential Evaluation
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The osteogenic and adipogenic differentiation capacity of MSC was assessed at early passages by incubating cells with α‐MEM (Gibco, Life Technologies), 10% FBS (Mesenchymal Stem Cell Stimulation Supplement, StemCell Technologies), 1% Pen/Strep, and 2 mM l ‐Glu supplemented with 10−7mol/L dexamethasone, 50 mg/ml l ‐ascorbic acid; starting from day +7 of the culture 5 mmol/L β‐glycerol phosphate (all from Sigma‐Aldrich) was added to the medium. Adipogenic differentiation was evaluated at the same passages by incubating cells with α‐MEM, 10% FBS, 1% Pen/Strep, and 2 mM l ‐Glu supplemented with 10‐7mol/L dexamethasone, 50 mg/ml l ‐ascorbic acid, 100 mg/ml insulin, 50 mmol/L isobutyl methylxanthine, 0.5 mmol/L indomethacin, and 5 mmol/L β‐glycerol phosphate (all from Sigma‐Aldrich). Both osteogenic and adipogenic cultures were incubated for at least 3 weeks before evaluating differentiation. To detect osteogenic differentiation, cells were stained for calcium depositions with Alizarin Red S (Sigma‐Aldrich).
Adipogenic differentiation was evaluated through the morphological appearance of fat droplets stained with Oil Red O (Bio‐Optica). Differentiation capacity was evaluated by RT‐qPCR for the expression of adipogenic (PPARγ, FABP4, LPL) and osteogenic genes (RUNX2, SPARC, COL1A2).
Adipogenic differentiation was evaluated through the morphological appearance of fat droplets stained with Oil Red O (Bio‐Optica). Differentiation capacity was evaluated by RT‐qPCR for the expression of adipogenic (PPARγ, FABP4, LPL) and osteogenic genes (RUNX2, SPARC, COL1A2).
4
Osteogenic Differentiation of hASCs
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The isolation of hASCs has been described earlier (6 (link)).
Pooled hASCs from 3 donors at passage 3 were used. hASCs
were either or not incubated with 10-8 M 1,25-(OH)2VitD3at room temperature for 30 minutes. Then, the cells were
washed twice with PBS to remove 1,25-(OH)2VitD3,
centrifuged, and resuspended in Dulbecco’s Minimum
Essential Medium (DMEM, Gibco, Life Technologies,
USA) without any supplements. Cells were seeded at
the density of 5.5×104 cells per 25-35 mg of BCP20/80
scaffold in 2 mL tubes (Eppendorf Biopur®, Germany),
and allowed to adhere for 30 minutes to the scaffolds.
After washing twice with PBS, scaffolds with attached
cells were transferred into 12-well plates with Costar®
Transwell® containers (Corning Life Sciences, Lowell,
MA, USA) containing expansion medium (DMEM)
supplemented with 10% fetal clone I (FCI, ThermoFisher
Scientific, USA) as an alternative to fetal bovine serum
(FBS), antibiotics [1% penicillin/streptomycin/fungizone
(PSF)), 50 μM ascorbic acid (Merck, Germany), and
10 mM β-glycerol phosphate (Merck, Germany). The
hASCs-seeded scaffolds were incubated at 5% CO2 in a
humidified incubator at 37˚C for 3 weeks.
Pooled hASCs from 3 donors at passage 3 were used. hASCs
were either or not incubated with 10-8 M 1,25-(OH)2VitD3at room temperature for 30 minutes. Then, the cells were
washed twice with PBS to remove 1,25-(OH)2VitD3,
centrifuged, and resuspended in Dulbecco’s Minimum
Essential Medium (DMEM, Gibco, Life Technologies,
USA) without any supplements. Cells were seeded at
the density of 5.5×104 cells per 25-35 mg of BCP20/80
scaffold in 2 mL tubes (Eppendorf Biopur®, Germany),
and allowed to adhere for 30 minutes to the scaffolds.
After washing twice with PBS, scaffolds with attached
cells were transferred into 12-well plates with Costar®
Transwell® containers (Corning Life Sciences, Lowell,
MA, USA) containing expansion medium (DMEM)
supplemented with 10% fetal clone I (FCI, ThermoFisher
Scientific, USA) as an alternative to fetal bovine serum
(FBS), antibiotics [1% penicillin/streptomycin/fungizone
(PSF)), 50 μM ascorbic acid (Merck, Germany), and
10 mM β-glycerol phosphate (Merck, Germany). The
hASCs-seeded scaffolds were incubated at 5% CO2 in a
humidified incubator at 37˚C for 3 weeks.
5
Human Osteoblast Cultivation on SAE-Ti
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Human osteoblasts (HOb) derived from healthy bone (Cell Applications Inc., San Diego, CA) were expanded in a proliferation medium. The medium was prepared by supplementing low‐glucose Dulbecco's Modified Eagle's Medium with 0.584 g L−1l ‐glutamine (DMEM; Merck, Darmstadt, Germany), foetal bovine serum (10%; FBS; Merck), and penicillin/streptomycin (100 U ml−1; Gibco, Thermo Fisher Scientific, Waltham, MA). FBS was used instead of human serum aiming to distinguish between the human proteins produced by the cells and the bovine proteins adsorbed onto the surface. The osteoblasts were seeded at the density of 2.5 × 104 per cm2 on the SAE‐Ti samples in a 48‐well plate with osteogenic medium (DMEM, 1% penicillin/streptomycin, 10% FBS, 1% ascorbic acid [5 μg ml−1; Merck] and β‐glycerol phosphate [100 mM; Merck]). The cells were cultured for 1, 3, and 7 days in a humidified atmosphere (37°C, 5% CO2). Cells cultured in polystyrene wells without Ti discs were used as control (C). The cell culture medium was changed every 3 days.
6
Osteogenic Differentiation Protocol
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For osteogenic differentiation, 1 × 105 cells/well were seeded into a 6-well plate and incubated at 5% CO2 and 37 °C for 1 day. On the next day, media were exchanged. Control cells received the cell-line-specific medium, while the tested cells got osteogenic differentiation medium consisting of DMEM (Pan Biotech, Aidenbach, Germany) supplemented with 10% FCS (FCS; Biochrom GmbH, Berlin, Germany), 100 U/mL penicillin, 0.1 mg/mL streptomycin (Sigma-Aldrich, Taufkirchen, Germany), 100 nM dexamethasone 21-phosphate disodium salt (Sigma-Aldrich, Taufkirchen, Germany), 10 mM β-glycerol phosphate (Merck, Millipore, Darmstadt, Germany) and 50 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, Taufkirchen, Germany). The medium was replaced two times per week, and cells were cultured for 21 days at 5% CO2 and 37 °C. Afterward, cells were fixed in 4% paraformaldehyde (Thermo Scientific, Waltham, MA) and stained with Alizarin Red S (Sigma-Aldrich, Taufkirchen, Germany) for 30 min at RT and washed 3 times with aqua destillata (A. dest.).
7
Coculture Dynamics of Bone and Endothelial Cells
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BMSC and HUVEC were cocultured in medium Complete EGM2 at following densities: 50,000 cells/well of 24 well plate (for alizarin Red S staining and immunohistochemistry (IHC)); 300,000 cells/well of 6 well plate for quantitative polymerase chain reaction (qPCR); 700,000 cells/10 cm dish for Enzyme-linked immunosorbent assay (ELISA). When the cells reached total confluence, they were switched to i) osteogenic medium (OM) which is EGM2 containing 2% FBS, 50 μg/mL ascorbic acid (Sigma, Schnelldorf, Germany)), 10mM β glycerolphosphate (Merck, Schnelldorf, Germany)) and 10−7 M dexamethasone (Sigma, Schnelldorf, Germany)); ii) OM added 100 100 pg/mL IL-1β; iii) OM supplemented with 100 μM COCl2, iv) OM added 0.5 mM DMOG and v) OM in 1% O2. The addition of IL-1β was to evaluate osteogenesis in inflammatory conditions while the supplement of COCl2 and DMOG was to examine osteogenic differentiation in hypoxic conditions. Moreover, EGM2 supplemented with 2% FBS only was used as a control (CTR) medium. The media were freshly changed twice per week. The experiment lasted for 9 days.
8
Osteogenic Differentiation with IL1-β
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To stimulate osteogenic differentiation, cells were firstly cultured in complete medium supplemented with FGF-2 at 5 × 104 cells/well on 24-well plates or 3 × 105 cells/well on 6-well plates for Alizarin Red S stain and qPCR, respectively. For Alizarin Red S stain, the experiment was carried out in triplicate. When the cells reached confluence, the medium was switched to osteogenic medium (OM) which is complete medium containing 50 μg/ml ascorbic acid (Sigma), 10−7 M dexamethasone (Sigma), and 10 mM β glycerolphosphate (Merck). Moreover, 50 pg/ml IL1-β (R&D Systems) was also added to the OM. The concentration of IL1-β used for this experiment procedure was inferred from literature data [16 (link)–18 (link)]. The complete medium was used as control. OM and OM supplemented with IL1-β were prepared freshly and added to the cells twice per week. The differentiation was prolonged for 3 weeks.
9
Murine Osteoblast and Osteocyte Culture
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Murine osteoblastic cell line from ATCC (cat. CRL-2593), MC3T3-E1, were seeded in High-Glucose DMEM, (Euroclone, Milan Italy), supplemented with 10% FBS (Euroclone, Milan, Italy), as previously reported [34 (link)]. We replaced culture medium twice a week and MC3T3-E1 cells were trypsinized weekly. Murine osteocyte-like cells, MLO-Y4, were kindly provided by Dr. Romanello (Hospital “Santa Maria della Misericordia”, Udine, Italy). We coated plates of 10 mm with type I collagen. MLOY-4 cells were seeded as previously described. The cells were trypsinized twice a week.
To study MC3T3-E1 differentiation, cells were maintained as previously described [40 (link)]. In brief, cells were seeded in medium supplemented withl -ascorbic acid 50 μg/mL and β-glycerolphosphate (10 mM, both reagents from Merck KGaA (Darmstadt, Germany). Cultures were maintained for 7 days before experiments. All the experiments with steroids were performed in medium without phenol red and charcoaled FBS.
To study MC3T3-E1 differentiation, cells were maintained as previously described [40 (link)]. In brief, cells were seeded in medium supplemented with
10
Culturing Human Osteoblasts on Titanium
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Human osteoblasts (HOb) cell line derived from healthy bone (Cell Applications Inc., San Diego, CA, USA) were expanded in a low-glucose Dulbecco′s Modified Eagle′s Medium with 0.584 g L -1 L-glutamine (DMEM; Merck, Darmstadt, Germany), supplemented with foetal bovine serum (10%; FBS; Merck) and penicillin/streptomycin (100 U mL -1 ; Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Using FBS instead of human serum allows differentiating between human proteins produced by the cells and the bovine proteins from the serum. Cells were seeded at the density of 2.5 × 10 4 per cm 2 in empty wells or wells with Ti discs in 48-well plates. Half of the samples were cultured with non-supplemented (NM; DMEM, 1% penicillin/streptomycin, 10% FBS) and the other half with osteogenic medium (OM; DMEM, 1% penicillin/streptomycin, 10% FBS, 1% ascorbic acid (5 µg mL -1 ; Merck) and β-glycerol phosphate (100 mM; Merck)). The osteoblasts were cultured for 1 or 7 days in a humidified atmosphere (37 °C, 5% CO2), and the medium was changed every 3 days.
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