To test their adipogenic differentiation potential, MSCs were cultured in medium consisting of DMEM high glucose media with 10 % (v/v) FCS, 2 mM L-glutamine, 100 U/ml Pen/Strep, 1 mM dexamethason (Sigma), 0.5 mM 1-methyl-3-isobutylxanthin (Sigma) and 10 mg/ml Insulin (Sigma). Medium was changed twice a week. Osteogenic differentiation was induced using DMEM low glucose with 10 % (v/v) FCS, 2 mM L-glutamine, 100 U/ml Pen/Strep, 100 nM dexamethason (Sigma), 200 mM L-ascorbic acid 2-phosphate (Sigma) and 10 mM B-glycerophosphate (Sigma). After 3 weeks the cells were stained with Oil red O (Sigma) for adipogenic and Alizarin red S (Sigma) for osteogenic differentiation. To check for the MSC-specific immunophenotype, surface marker expression was tested using the Stemflow hMSC Analysis Kit (BD Biosciences).
Dexamethason
Manufactured by Merck Group
Sourced in China
Dexamethason is a synthetic glucocorticoid medication used in laboratories for various research and experimental purposes. It is a potent anti-inflammatory and immunosuppressant agent. The core function of Dexamethason is to modulate and regulate biological processes in cell culture and animal studies.
Automatically generated - may contain errors
Lab products found in correlation
β glycerophosphate, by Merck Group (4 mentions)
Insulin, by Merck Group (3 mentions)
Oil red o, by Merck Group (3 mentions)
Alizarin red s, by Merck Group (2 mentions)
L ascorbic acid 2 phosphate, by Merck Group (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (2 mentions)
Pen strep, by Merck Group (1 mentions)
Stemflow hmsc analysis kit, by BD (1 mentions)
Bcip nbt alkaline phosphatase color development kit, by Beyotime (1 mentions)
Microplate reader, by Tecan (1 mentions)
Inverted microscope, by Zeiss (1 mentions)
Alkaline phosphatase assay kit, by Beyotime (1 mentions)
Vitamin c, by Solarbio (1 mentions)
Cetylpyridine chloride, by Merck Group (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Fuchsin substrate chromogen, by Agilent Technologies (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Solarbio (1 mentions)
Oil red o staining kit, by Beyotime (1 mentions)
15 protocols using dexamethason
1
Evaluating Adipogenic and Osteogenic Potential of MSCs
2
Osteogenic Differentiation Assay Protocol
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The osteogenic-inducing medium was α-MEM supplemented with 10% FBS, 1% penicillin/streptomycin, varying concentrations of NAC, 10 mM Na-β-glycerophosphate (Solarbio, Beijing, China), 100 nM dexamethason (Sigma-Aldrich, MO, USA) and 50 μg/mL vitamin C (Solarbio, Beijing, China). Alkaline phosphatase (ALP) assays were conducted after 5-day osteogenic induction. ALP staining was carried out using a BCIP/NBT alkaline phosphatase color development kit (Beyotime, Shanghai, China). ALP activity was performed using alkaline phosphatase assay kit (Beyotime, Shanghai, China). Protein concentration of each sample was as described above. Alizarin red S (ARS) staining was performed after 2-week osteogenic induction using 0.2% ARS (Solarbio, Beijing, China) at 37 °C for 30 min. For semi-quantitative analysis, PBS containing 10% cetylpyridine chloride (Sigma-Aldrich, MO, USA) was added into each well to dissolve precipitation after staining and the absorbance was measured using a microplate reader (Tecan, Männedorf, Switzerland) at 542 nm. Cells were observed using an inverted microscope (Zeiss, Oberkochen, Germany).
3
Isolation of Primary Human Osteoblasts
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Isolation of human primary osteoblasts followed the procedure described previously by Lochner et al. [38 (link)]. Briefly, cells were isolated from the spongiosa of the femoral heads of patients undergoing primary total hip replacement. The samples were collected with patient agreement and approval by the Local Ethical Committee (registration number: A 2010-10). Cultivation was done in osteogenic cell culture medium (MEM Dulbecco, Biochrom AG, Berlin, Germany) with 10% FCS, 1% penicillin/streptomycin, 1% amphotericin B, 1% HEPES buffer (all: Gibco®-Invitrogen, Darmstadt, Germany) including osteogenic additives (dexamethason (100 mM), L-ascorbic acid (50 mg/mL) and β-glycerophosphate (10 mM) (all from Sigma-Aldrich, Munich, Germany)). Alkaline phosphatase staining with fuchsin substrate chromogen (DAKO, Hamburg, Germany) was done to verify the osteogenic character of the isolated cells. Cultivation was carried out in an incubator (Binder GmbH, Tuttlingen, Germany) at simulated in vivo conditions 37 °C, 5% CO2 and 21% O2 for one week.
A total of seventeen different donors (10 ♂, 7 ♀) were used for all experiments. The average age was 66 ± 9.4 years. Cells were not pooled; one donor was used for one scaffold per experiment.
A total of seventeen different donors (10 ♂, 7 ♀) were used for all experiments. The average age was 66 ± 9.4 years. Cells were not pooled; one donor was used for one scaffold per experiment.
4
Adipogenic Differentiation Protocol
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The adipogenic-inducing medium was α-MEM supplemented with 10% FBS, 1% penicillin/streptomycin, 111 μg/mL IBMX (Solarbio, Beijing, China), 72 μg/mL indomethacin (Solarbio, Beijing, China), 5 μg/mL insulin (Aladdin Chemical, Shanghai, China) and 0.4 μg/mL dexamethason (Sigma-Aldrich, MO, USA). Oil red o staining after 15-day induction was carried out using a modified oil red o staining kit (Beyotime, Shanghai, China).
5
Adipogenic Differentiation Assay Protocol
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Cells were plated at a density of 5 × 104 per well in 24-well plates. After overnight incubation, adipogenic induction medium with 5 μg/ml insulin (SIGMA), 1 μM Dexamethason (SIGMA), 500 μM 3-isobutyl-1-methylxanthine (IBMX), 1 μg/ml rosiglitazone (ENZO Lifesciences) in DMEM-F12 was applied for 2 days. Subsequently, cells were cultured for another 2 days in DMEM-F12 with 5 μg/ml insulin, 10% FBS, 1% streptomycin/penicillin and for 2 more days in the medium without insulin. For Oil Red O staining, cells were rinsed twice with PBS and fixed with 10% formalin in PBS for 5 min at RT. Cells were subsequently fixed for 1 h at RT with fresh formalin solution, washed with 60% isopropanol and then completely dried. Oil Red O working solution, made up of 6 parts filtered Oil Red O stock (0.7 g Oil Red O in 200 ml isopropanol; Sigma) and 4 parts distilled water, was applied to the dried well for 10 min incubation. Cells were rinsed with distilled water 4 times and dried prior to microscopic observation.
6
Immortalized Hepatocyte Culture Protocol
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Immortalized embryonic hepatocytes were cultured in Medium 199 (WelGENE) that was supplemented with 10% fetal bovine serum (WelGENE) and 1% penicillin-streptomycin (WelGENE), as previously described (Back et al., 2009 (link)). AML12 muse normal hepatocytes were obtained from the American Type Culture Collection (ATCC, USA). The cells were cultured in DMEM/F12 (WelGENE) that was supplemented with 10% fetal bovine serum (WelGENE), 100 nM dexamethason (Sigma-Aldrich), 1% insulin-transferrin-selenium-pyruvate supplement (ITSP; WelGENE), and 1% penicillin-streptomycin (WelGENE). The Lenti-X 293T Cell Line (Clontech, USA) was cultured in DMEM (WelGENE) containing 10% fetal bovine serum (WelGENE) and 1% penicillin-streptomycin (WelGENE).
7
Osteogenic and Adipogenic Differentiation of BMSCs
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Cells were identified with alizarin red staining for osteogenic differentiation and Oil
Red O staining for adipogenic differentiation. To induce osteogenic differentiation, BMSCs
were grown in osteogenic-inducing medium α-MEM containing 10% FBS, 100 U/ml penicillin,
100 μg/ml streptomycin sulfate, 50 μg/ml ascorbic acid (Sigma, St. Louis, MO, USA), 5 mM
β-glycerol phosphate (Sigma) and 10-8 M dexamethason (Sigma) for 21 days. After
that, cells were fixed in 4% paraformaldehyde for 30 min, followed by two washes with
ice-cold PBS, were stained for 30 min with alizarin red, and then underwent two washes as
previously described. The secreted osteocalcin level was then explored. To induce
adipogenic differentiation, cells were cultured in the adipogenic differentiation medium,
consisting of α-MEM supplemented with 10% FBS, 1 μmol/l dexamethasone, 0.05 mmol/l
3-isobutyl-1-methylxanthine, 10 μmol/ml bovine insulin and 200 μmol/l indometacin for 7
days. Oil Red O staining was performed to detect the lipid droplets. Briefly, cultured
BMSCs were fixed in 4% paraformaldehyde for 30 min and washed with 60% isopropanol. They
were stained with Oil Red O for 5 min, they then underwent a rinse with 60% isopropanol
and two further rinses with ice-cold PBS.
Red O staining for adipogenic differentiation. To induce osteogenic differentiation, BMSCs
were grown in osteogenic-inducing medium α-MEM containing 10% FBS, 100 U/ml penicillin,
100 μg/ml streptomycin sulfate, 50 μg/ml ascorbic acid (Sigma, St. Louis, MO, USA), 5 mM
β-glycerol phosphate (Sigma) and 10-8 M dexamethason (Sigma) for 21 days. After
that, cells were fixed in 4% paraformaldehyde for 30 min, followed by two washes with
ice-cold PBS, were stained for 30 min with alizarin red, and then underwent two washes as
previously described. The secreted osteocalcin level was then explored. To induce
adipogenic differentiation, cells were cultured in the adipogenic differentiation medium,
consisting of α-MEM supplemented with 10% FBS, 1 μmol/l dexamethasone, 0.05 mmol/l
3-isobutyl-1-methylxanthine, 10 μmol/ml bovine insulin and 200 μmol/l indometacin for 7
days. Oil Red O staining was performed to detect the lipid droplets. Briefly, cultured
BMSCs were fixed in 4% paraformaldehyde for 30 min and washed with 60% isopropanol. They
were stained with Oil Red O for 5 min, they then underwent a rinse with 60% isopropanol
and two further rinses with ice-cold PBS.
8
Characterization of hPDLCs Differentiation
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After defrosting, the morphology of hPDLCs was observed by an inverse phase contrast microscope (Leica DMIL, Germany) at ×10 magnification. The self-renewal and osteogenic ability of hPDLCs from the 4th passage were characterized by colony-forming unit (CFU) efficiency, ALP activity, and mineralization ability (Von-Kossa staining). CFU assay was performed on day 10 in proliferation medium. ALP activity of hPDLCs was measured on day 8 in osteogenic medium (proliferation medium containing 50 μg/mL ascorbic acid, 10 mM sodiumβ-glycerophosphate, and 10 nM dexamethason, all from Sigma). Human fibroblasts from foreskin (a kind gift from Department of Orthodontics and Oral Biology, Radboud University Medical Centre) and hBMSCs were used as negative and positive control for ALP activity assay. Von-Kossa staining was performed after 30 days of culture in osteogenic medium. All the assays are described in detail below.
9
Osteogenic Differentiation of Rat MSCs
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Rat MSCs were seeded with a density of 1 × 104 cells/cm2 and incubated overnight. Then, the medium was replaced with osteogenic medium consists of DMEM containing 10% FBS, antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin sulfate), 100 nM Dexamethason (Sigma, Saint Louis), 10 mM β-glycerophosphate (Sigma) and 50 μM L-ascorbate-2-phosphate (Sigma) in a humidified CO2 incubator at 37 °C. When the culture is 80–90% confluence, the cells were passaged at the ratio of 1:4 in 75-cm2 tissue culture flasks. Cells from the 3th to the 4th passage were used for this study.
10
Porcine Mesenchymal Stem Cell Adipogenesis
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Porcine mesenchymal stem cells isolated from bone marrow (BM) of Polish Large White pig were cultured in DMEM medium (Gibco) supplemented with 10 % FCS (Sigma), 5 ng/ml FGF-2 (PromoKine), 1 × NonEssential amino acids (Gibco), 2 mM L-Glutamine (PAA), 1 mM 2-Mercaptoethanol (Sigma) and a mixture of antibiotics (100 U/ml of Penicillin, 100 μg/ml of Streptomycin, Sigma) at 37 °C in 5 % CO2. In all experiments pMSC were used at early passages (P4-P8). To induce adipogenic differentiation, pMSCs were grown to confluency and were cultured with adipogenic differentiation medium composed of the basal medium supplemented with 50 μM IBMX (Sigma), 1 μM Dexamethason (Sigma-Aldrich), 100 μM Indomethacin (Sigma-Aldrich), 1 × ITS and 1 × Linoleic Acid (Sigma -Aldrich) and FGF-2 (5 μg/ml)). The cells were cultured for 7 days and adipocyte differentiation was monitored by phase contrast microscopy.
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