We purchased Whatman 114 filter paper, glycerol-2-phosphate, ascorbic acid, alizarin red S and 4,6-diamidino-2-phenylindole (DAPI) from Sigma-Aldrich (St Louis, MO). We obtained the paraformaldehyde solution (16% (v/v)) from Electron Microscopy Sciences (Hatfield, PA). Mouse antibodies to Osteocalcin and Alexa 647-labeled antibodies to rabbit IgG were supplied by Abcam (Cambridge, MA). We purchased trypsin-EDTA, penicillin-streptomycin, phalloidin (Texas Red-X), fetal calf serum (FCS), fetal bovine serum (FBS), alpha-minimal essential medium (α-MEM) medium, and Dulbecco’s phosphate buffered saline (DPBS) from Invitrogen (Carlsbad, CA). We bought the OsteoImage kit from Lonza Walkersville Inc. (Walkersville, MD). We used all of the reagents as received without further purification.
Glycerol 2 phosphate
Manufactured by Merck Group
Sourced in United States
Glycerol-2-phosphate is a chemical compound that functions as an intermediate in the metabolism of glycerol. It is commonly used in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (28 mentions)
Ascorbic acid, by Merck Group (14 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (8 mentions)
Indomethacin, by Merck Group (7 mentions)
Insulin, by Merck Group (7 mentions)
L ascorbic acid, by Merck Group (6 mentions)
Ascorbic acid 2 phosphate, by Merck Group (6 mentions)
L ascorbic acid 2 phosphate, by Merck Group (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
α mem, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Oil red o, by Merck Group (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Alizarin red s, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (2 mentions)
Cholecalciferol, by Merck Group (2 mentions)
3 isobutyl 1 methylxanthine ibmx, by Merck Group (2 mentions)
Silver nitrate, by Merck Group (2 mentions)
40 protocols using glycerol 2 phosphate
1
Osteogenic Differentiation Assay
2
Osteogenic Differentiation of iPSCs
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iPSCs were differentiated into osteogenic lineages using previously published protocols [24 (link)]. Briefly, 2 × 105 iPSCs were maintained in feeder-free conditions and differentiation was induced in gelatin-coated 24-well plates with 20% mTeSR1 medium (StemCell Technologies, Vancouver, BC, CA) and 80% osteogenic differentiation medium composed of KnockOut DMEM (Gibco) supplemented with 20% FBS (Gibco), 1% non-essential amino acids (Wako), 0.1 mM 2-mercaptoethanol (Sigma), 2 mM Gluta-MAX (Wako), 10 mM glycerol-2-phosphate (Sigma), 1 nM dexamethasone (Sigma), 50 µg/mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma), and 1 µM all-trans-retinoic acid (Sigma), and 10 µM rock inhibitor Y-27632 (Wako). On day 2, the medium was replaced with 100% osteogenic differentiation medium without Y-27632 and changed on days 4 and 7.
3
Osteogenic Differentiation of hMSCs
4
Comprehensive Ubiquitin Assay Protocol
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Hypoxanthine, sorbitol, percoll, MG132, Epoxomicin, Lactacystin, Clasto-Lactacystin β-lactone, Gliotoxin, MG115, EDTA, sodium phosphate dibasic, monosodium phosphate, Tris HCl, sodium chloride, Tween-20, sodium pyrophosphate, glycerol-2-phosphate, saponin, bovine serum albumin (BSA), dithiothreitol (DTT) and phenylmethylsulphonyl fluoride (PMSF) were purchased from Sigma. Glutathione acceptor beads, protein A donor beads, DELFIA enhancement solution and DELFIA secondary antibody (Eu-N1 rabbit Anti-mouse-IgG) came from Perkin Elmer. NP-40 was purchased from Calbiochem, sodium fluoride from Panreac, antibody anti-ubiquitin P4D1 from Santacruz, deubiquitylases inhibitor PR-619 came from Merck, complete mini EDTA protease inhibitor cocktail from Roche, antibody anti-ubiquitin FK2 from Enzo, biotin-TUBEs from Life sensor, RPMI 1640 medium from Gibco, AlbuMAX II from Invitrogen, bortezomib from Selleckchem, enhanced chemiluminescence (ECL) from GE Healthcare and PBS from Oxoid. Atovaquone was prepared in house.
5
Osteogenic Differentiation of Mesenchymal Cells
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Bovine fibrinogen, thrombin, aminocaproic acid, L-Ascorbic acid and glycerol 2-phosphate were purchased from Sigma (St. Louis, MO). Rat tail type I collagen was purchased from BD Bioscience (San Jose, CA). Fetal bovine serum (FBS), HEPES buffer solution, and penicillin-streptomycin solution were from GibcoBRL (Carlsbad, CA), and ascorbic acid-free α-MEM was from WelGene (Daegu, Korea). The MicroBCA assay kit was from Pierce-Thermo (Rockford, IL). Qunat-iT PicoGreen dsDNA-assay kit was from Invitrogen (Eugene, OR). West-Zol was from Intron Biotechnology (Seoul, Korea). A Dual-GloTM Luciferase Assay System was from Promega (Madison, WI). Protease inhibitor cocktail tablets (Complete) were from Roche (Basel, Switzerland). Anti-Runx2, anti-actin, and HRP-conjugated IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-fibronectin and anti-vitronectin antibodies were from Chemicon (Temecula, CA).
6
Osteogenic Differentiation of Human MSCs
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The HEK293 cells were purchased from ATCC and cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin/neomycin (PSN). The human BM-MSCs were isolated from bone marrow, which aspirated from healthy donors, and then cultured and characterized using flow cytometry for MSC phenotypic markers like CD3-PE, CD16-FITC, CD19-FITC, CD33-FITC, CD34-PE, CD38-FITC, CD45-FITC, and CD133-PE. Isolated human BM-MSCs were cultured in Minimum Essential Medium Alpha (MEMα) plus 10% heat-inactivated FBS and 1% PSN. To initiate osteogenic differentiation of human MSCs, they were seeded into a 12-well plate at a density of 2 × 105 cells per well; then the osteo-differentiation was induced by addition of 10 nM dexamethasone (Sigma-Aldrich, USA), 50 μg/mL ascorbic acid 2-phosphate (Sigma-Aldrich, USA), and 10 mM glycerol 2-phosphate (Sigma-Aldrich, USA) into the culture medium. The differentiation medium was changed every 3 days.
7
Osteogenic Differentiation of C3H10T1/2 Murine MSCs
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C3H10T1/2, murine mesenchymal stem cells, were purchased from the Korean Cell Line Bank (KCLB) affiliated to Seoul National University Hospital (SNUH). C3H10T1/2 cells with passage number 26 were plated to the 18 × 18 mm GO/Glass slides at a density of 5 × 104 cells per slide and cultured with Dulbecco’s Modified Eagle Medium (DMEM; GIBCO, USA). The medium was supplemented with 10% fetal bovine serum (FBS; GIBCO, USA) and 1% penicillin/streptomycin (Pen/Strep; GIBCO, USA). For osteogenic differentiation, cells were influenced by osteogenic differentiation substrate, as cultured in DMEM supplemented with 50 mg/ml L-ascorbic acid (Sigma-Aldrich, USA), 1% Pen-Strep, 10% FBS, 100 nM dexamethasone (Sigma-Aldrich, USA), and 10 mM glycerol-2-phosphate (Sigma-Aldrich, USA), for 14 days. Osteogenic differentiation medium was replaced for every day.
8
Isolation and Culture of Rat Calvarial Cells
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Rat calvaria were collected from 3-day-old Sprague-Dawley rats. All procedures involving animals were performed following the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of CHA University (IACUC 190076, March, 2019). After removal of sutures and adherent mesenchymal tissues and surgical isolation from the skull, the calvaria were introduced with 5 sequential (10–15 min) digestions at 37 °C in a solution containing 0.1% dispase and 0.1% collagenase P. Cells released from the second to the fifth digestions were centrifuged, collected, and resuspended. Those cells were cultured and incubated in proliferation medium (DMEM (Gibco BRL) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco BRL) and 100 units/mL penicillin (GibcoBRL)) or osteogenic medium (DMEM supplemented with 10% (v/v) FBS, 0.2 mM ascorbic acid (Sigma, St. Louis, MO, USA)), 1% GlutaMAX (GibcoBRL), 10 mM glycerol 2-phosphate (Sigma), and 100 units/mL penicillin (GibcoBRL) in humidified air with 5% (v/v) CO2 at 37 °C until the cells reached confluency.
9
Differentiation of BM-MSCs into Osteoblasts and Adipocytes
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BM-MSCs were induced to differentiate into osteoblasts and adipocytes using the following procedure. The induction medium for osteogenesis was Gibco® Iscove's modified Dulbecco's medium (IMDM; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 0.1 µM dexamethasone, 0.2 mM ascorbic acid 2-phosphate and 10 mM glycerol 2-phosphate (Sigma-Aldrich; Merck KGaA). The induction medium for adipogenesis was IMDM supplemented with 10% FBS, 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 10 µg/ml insulin and 60 µM indomethacin (Sigma-Aldrich; Merck KGaA). After 3 days, the culture medium was completely replaced and the medium was subsequently changed twice weekly thereafter. After the predetermined culture time had elapsed, adipocytes were stained with Oil Red O at room temperature for 30 min; the osteoblasts were identified using alkaline phosphatase (ALP) and Alizarin Red S assays were performed at room temperature in the dark for 50 min (Sigma-Aldrich; Merck KGaA).
10
Adipogenic and Osteogenic Differentiation of UCB-MSCs
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UCB-MSCs were seeded into 6-well plates (Corning Inc., Corning, NY) at 2 × 104 cells/well, in adipogenic induction medium containing 10% fetal bovine serum (Gibco), 1 μM of dexamethasone, 100 μM of indomethacin, 500 μM of 3-isobutyl-1-methylxanthine, and 10 μg/mL of insulin (all from Sigma-Aldrich, St Louis, MO). The fresh medium was replaced every 3 days. After 21 days, Oil Red-O (Sigma-Aldrich) staining was used to identify intracellular accumulation of lipid-rich vacuoles. Briefly, cells were fixed with 4% paraformaldehyde for 30 minutes, washed with phosphate buffered saline, and stained with a working solution of 0.3% Oil Red-O in phosphate buffered saline for 20 minutes. For osteogenesis, after preparation with 0.1 µM of dexamethasone (Sigma-Aldrich), 0.2 µM of ascorbic acid 2-phosphate (Sigma-Aldrich), 10 mM of glycerol 2-phosphate (Sigma-Aldrich), and 10% fetal bovine serum, and fixation cells were stained with Alizarin red S (Fluka Buchs SG, Switzerland) [27 (link)].
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