Ten percent GelMA solutions were prepared as described earlier, and hASCs were uniformly suspended in this solution at 10 million cells/mL. For sample groups with loaded growth factor, BMP-2 was mixed into the prepolymer solution at the concentrations indicated in Table I . BMP-2 reconstitution buffer was added to groups that received less or no BMP-2 solution to ensure all groups had the same ratio of GelMA to BMP-2 buffer. Hydrogels were formed by crosslinking the cell- and BMP-2-laden GelMA solutions as described above and then placed in 24-well culture plates and cultured in DMEM-LG (Sigma Aldrich) containing 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, 10 mM β-glycerophosphate disodium salt (EMD Millipore, Billerica, MA) and 120 nM L-ascorbic acid phosphate magnesium salt (Wako Chemicals, Osaka, Japan) in a humidified 37°C, 5% CO2 incubator. In the conditions receiving exogenous growth factor supplementation, BMP-2 was added to the media at the concentrations presented in Table I . Control hydrogels were made without cells and cultured under the same conditions to measure any calcification that was not cell-mediated. For all groups, culture medium was replaced every 3 days.
β glycerophosphate disodium salt
Manufactured by Merck Group
Sourced in United States
β-glycerophosphate disodium salt is a chemical compound used in various laboratory applications. It serves as a source of phosphate ions and is often utilized in cell culture media, buffers, and other research applications requiring a phosphate source.
Automatically generated - may contain errors
Lab products found in correlation
Ascorbic acid, by Merck Group (3 mentions)
L ascorbic acid, by Merck Group (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
L ascorbic acid phosphate magnesium salt, by Fujifilm (1 mentions)
Dmem lg, by Merck Group (1 mentions)
Tween 80, by Fujifilm (1 mentions)
Nad nadh assay kit wst, by Dojindo Laboratories (1 mentions)
Sodium chloride (nacl), by Fujifilm (1 mentions)
Fastprep 24, by MP Biomedicals (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
H dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Beyotime (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
L proline, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Ab183797, by Abcam (1 mentions)
Ab6747, by Abcam (1 mentions)
13 protocols using β glycerophosphate disodium salt
1
BMP-2 Loaded GelMA Hydrogel Culture
2
Intracellular NAD+ and NADH Quantification
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Five grams of fermented sample at pH 5.5 was mixed with 1.7 mL of 1 M sodium citrate and 0.7 mL of saline solution consisting of 0.85% (wt/vol) NaCl (Fujifilm Wako), 0.5% (wt/vol) β-glycerophosphate disodium salt (EMD Millipore Corp.), and 0.1% (wt/vol) Tween 80 (Fujifilm Wako, pH 7.0). After vortexing and centrifugation at 4°C and 20,000 × g for 5 min, the cell pellet was resuspended in 10 mL of 50 mM potassium phosphate buffer and centrifuged again at 4°C and 20,000 × g for 5 min. The cell pellet was stored at -80°C until measurement. Intracellular NAD + and NADH concentration was measured using NAD/NADH Assay Kit-WST (Dojindo) with the addition of the cell-lysing process described below. The cell pellet was resuspended in 1.5 mL of 50 mM potassium phosphate buffer, and the volume containing 5.0 × 10 6 cfu was centrifuged at 4°C and 20,000 × g for 5 min. The cell pellet was resuspended in 600 µL of NAD-NADH extraction buffer in the kit and transferred to a screw-cap tube with 0.6 g of 0.1-mm zirconium beads. The cell was lysed in FastPrep-24 (MP Biomedicals) twice for 30 s at a speed of 6.5 m/s and centrifuged at 4°C and 20,000 × g for 5 min. The supernatant was deproteinated using a 10K molecular weight cutoff spin filter included in the kit, and NAD + and NADH concentrations in the filtered sample were measured according to the manufacturer's instruction.
3
Synthesis of Chitosan-Graphene Oxide Composite
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β-Glycerophosphate disodium salt was obtained from Sigma-Aldrich Co.(US). Medium molecular weight chitosan(molecular weight≈21,0000 Da, deacetylation degree≥95%) was purchased from Tokyo Chemical Industry Co., Ltd. Acetic acid (chemical reagents of analytical grade) was provided by Baoxin Bio-Technology Co., Ltd. (Chengdu, China). Single-layered graphene oxide, purity>99%, diameter of 0.5–5 μm, thickness of 0.8–1.2 nm was purchased from Nanjing/Jiangsu XFNANO Materials Tech Co., Ltd. (Nanjing, China).
4
Isolation and Culture of Rat Osteoblasts
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A total of 5 female Wistar rats (1-day-old) weight 5–6 g were purchased from Shandong University Laboratory Animal Center. The animal care and experiments were approved by the Ethics Committee for Animal Care and Use of Shandong Academy of Medical Sciences. Culture of fetal rat calvarial osteoblasts was performed as described previously (11 (link)). The growth medium was supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin-streptomycin (Beyotime, Haimen, China) in H-DMEM (Gibco, Waltham, MA, USA). The osteoblast differentiation medium (OBM) was supplemented with 10 mM β-glycerophosphate disodium salt hydrate and 50 µg/ml ascorbic acid (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for the growth medium.
5
Mesenchymal Stem Cell Culture Conditions
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Standard growth medium: low-glucose DMEM GlutaMAX, 15% fetal calf serum (FCS), penicillin 100 U/ml—streptomycin 100 µg/ml (Life Technologies, UK).
Osteogenic medium: low-glucose DMEM GlutaMAX, 15% FCS, penicillin 100 U/ml—streptomycin 100 µg/ml, 10 mM β-glycerophosphate disodium salt hydrate, 10 nM dexamethasone, 50 µg/ml L-ascorbic acid (Sigma-Aldrich, USA).
Chondrogenic medium: low-glucose DMEM GlutaMAX, 1% FCS, penicillin 100 U/ml—streptomycin 100 µg/ml, 1X ITX-G (Life Technologies, UK), 50 µg/ml L-proline, 100 nM dexamethasone, 50 µg/ml L-ascorbic acid (Sigma-Aldrich, USA), 10 ng/ml recombinant human TGFβ3 (Miltenyi Biotec, Germany).
Cells were grown in the atmosphere of 5% CO2 unless otherwise specified.
Osteogenic medium: low-glucose DMEM GlutaMAX, 15% FCS, penicillin 100 U/ml—streptomycin 100 µg/ml, 10 mM β-glycerophosphate disodium salt hydrate, 10 nM dexamethasone, 50 µg/ml L-ascorbic acid (Sigma-Aldrich, USA).
Chondrogenic medium: low-glucose DMEM GlutaMAX, 1% FCS, penicillin 100 U/ml—streptomycin 100 µg/ml, 1X ITX-G (Life Technologies, UK), 50 µg/ml L-proline, 100 nM dexamethasone, 50 µg/ml L-ascorbic acid (Sigma-Aldrich, USA), 10 ng/ml recombinant human TGFβ3 (Miltenyi Biotec, Germany).
Cells were grown in the atmosphere of 5% CO2 unless otherwise specified.
6
Osteogenic Differentiation Assay
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L-alanyl-L-glutamine and insulin-like growth factor-1 (IGF-1) were purchased from Thermo Fisher (Waltham, MA, USA); Lipofectamine 2000 reagent was obtained from Invitrogen (Carlsbad, CA, USA); β-glycerophosphate disodium salt, dexamethasone, and l-ascorbic acid were obtained from Sigma-Aldrich (St Louis, MO, USA); and primary antibodies against GRIA1 (ab183797), GAPDH (ab8245), and HRP secondary antibody (ab6747) were obtained from Abcam (Cambridge, MA, USA).
7
Osteogenic and Adipogenic Differentiation of BMSCs
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BMSCs were inoculated in 6-well dishes with 5 × 105 cells/well and cultured to 90% fusion in complete medium and then induced by osteogenic induction medium (complete medium supplemented with 100 nM dexamethasone, 10 mM β-Glycerophosphate disodium salt hydrate, 50 μM 2-Phospho-L-ascorbic acid trisodium salt (Sigma-Aldrich, USA)). The media were refreshed at 3-day intervals. After 21 days of osteogenic induction, the cells were fixed in 4% paraformaldehyde and then stained with Alizarin Red staining solution for 30 min at room temperature. To assess adipogenesis, cells were cultured in SD rat bone marrow mesenchymal stem cells adipogenic differentiation medium (Cyagen, China, RASMX-90031) following the manufacturer’s instructions. After 21 days of induction, cells were fixed in 4% paraformaldehyde for 30 min and were stained with Oil Red O for 30 min. The dishes were then washed twice with PBS and observed under a microscope [26 (link)].
8
Osteogenic Differentiation of MC3T3-E1 Cells
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MC3T3-E1 cells were plated in a 24-well plate at a density of 1 × 105 cells/well and incubated for 24 h. The medium was replaced with osteogenic differentiation medium (300 μL) supplemented with 10% FBS, ascorbic acid (50 μg/mL; Sigma-Aldrich), β-glycerophosphate disodium salt (10 mM; Sigma-Aldrich), and each BMP-2 complex (50 ng/mL BMP-2 and 500 μg/mL polymers). After 72 h of incubation, the cells were washed twice with phosphate-buffered saline (PBS). The cell lysate was prepared using radioimmunoprecipitation assay (RIPA) buffer (Fujifilm Wako Pure Chemical) containing a Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland). The enzymatic activity of ALP in the lysate was determined using LabAssay ALP (Fujifilm Wako Pure Chemical) according to the manufacturer’s instructions. The absorbance at 405 nm was measured on a Multiskan FC plate reader. The concentration of total protein in the cell lysates was determined using a Micro BCA Protein Assay Kit (Thermo Fischer Scientific) according to the manufacturer’s instruction. The activity of ALP was normalized to the amount of protein.
9
Synthesis of Chitosan-Based Biomaterials
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The β-glycerophosphate
disodium salt, Nar, and β-CD used in this study was acquired
from Sigma-Aldrich Co. (St. Louis, MO). The medium molecular weight
chitosan, with a molecular weight of approximately 210,000 Da and
a deacetylation degree of at least 95%, was obtained from Tokyo Chemical
Industry Co., Ltd. Acetic acid of analytical grade was provided by
Baoxin Bio-Technology Co., Ltd. (Chengdu, China). Unless otherwise
specified, all other chemicals were obtained from Invitrogen Life
Technologies (Carlsbad, CA).
disodium salt, Nar, and β-CD used in this study was acquired
from Sigma-Aldrich Co. (St. Louis, MO). The medium molecular weight
chitosan, with a molecular weight of approximately 210,000 Da and
a deacetylation degree of at least 95%, was obtained from Tokyo Chemical
Industry Co., Ltd. Acetic acid of analytical grade was provided by
Baoxin Bio-Technology Co., Ltd. (Chengdu, China). Unless otherwise
specified, all other chemicals were obtained from Invitrogen Life
Technologies (Carlsbad, CA).
10
WRN Protein Stabilization Assay
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Cells were cultured in a 225 cm2 flask (Falcon, 353138) to a maximum confluency of 95%, then cells were rinsed twice in PBS and lysed in 12.5 ml of lysis buffer per flask (50 mM Tris pH 7.8, 1% NP40 (Sigma-Aldrich, 74385), 120 mM NaCl (Sigma-Aldrich, 71380), 25 mM NaF (Merck, 1.06450.0025), 40 mM β-glycerophosphate disodium salt (Sigma-Aldrich, 50020), 100 µM sodium metavanadate (Sigma-Aldrich, 590088), 1 mM DL-DTT (Sigma-Aldrich, 43815), 100 µM phenylmethyl sulfonyl fluoride (Sigma-Aldrich, P-7626), 1 mM benzamidine (Sigma-Aldrich, B-6506), 1 µM microcystin (Alexis Biochemicals 350-012-M001)). The lysates were centrifuged at 4 °C for 10 min and total protein was quantified and adjusted to 0.5 µg µl−1. Then, 100 µl of cell lysates was then plated in 96-well plates, the lysates were treated with HRO761 starting at 10 µM using the HP Dispenser (software D300eControl) and then incubated for 72 h at 20 °C. After incubation, the plates were used for in WRN enzyme-linked immunosorbent assay (ELISA) analysis as described below. PS50 values are the levels of HRO761 needed to stabilize the WRN protein 50% over DMSO control.
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