Pre-adipocyte differentiation into mature adipocytes was achieved by exposing pre-adipocytes to an appropriate adipogenic hormone cocktail (dexamethazone, 3-isobutyl-1-methylxanthine, and insulin), according to a standard differentiation protocol as described elsewhere [38 (link)] In brief, two days past 70% confluency (day 0), cells were treated with 0.5 mM 3-isobutyl-1-methylxanthine (Sigma, Germany), 1.0 μΜ dexamethazone (Sigma, Steinheim, Germany), and 10 ng/mL of insulin, as final concentrations in DMEM. Two days later (day 2), the cells were cultured in maintenance medium, treated only with insulin (main adipogenic factor) and the medium was serially changed every two days, whereas lipid formation was monitored on a daily basis. All experiments were performed on the 8th day of the differentiation process.
Dexamethazone
Manufactured by Merck Group
Sourced in Germany
Dexamethasone is a synthetic glucocorticoid medication used as a potent anti-inflammatory and immunosuppressant. It is primarily used in laboratory research applications to study cellular processes and immune system functions.
Automatically generated - may contain errors
Lab products found in correlation
Insulin, by Merck Group (3 mentions)
Pioglitazone, by Fujifilm (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ab15160, by Abcam (1 mentions)
Recombinant proteins, by R&D Systems (1 mentions)
Ab76774, by Abcam (1 mentions)
Vitronectin, by Thermo Fisher Scientific (1 mentions)
3 isobutyl 1 methylxanthine ibmx, by Fujifilm (1 mentions)
Rosiglitazone, by Merck Group (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
Collagenase 1, by Merck Group (1 mentions)
Lactacystin, by Merck Group (1 mentions)
Forskolin, by Merck Group (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Anti actin antibody, by Abcam (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Fujifilm (1 mentions)
Mg132, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Anti gfp, by Abcam (1 mentions)
6 protocols using dexamethazone
1
Adipocyte Differentiation from Pre-adipocytes
2
Intestinal Organoid Culture Reagents
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The recombinant proteins and neutralizing antibodies for mouse TNF and IL-6 were obtained from R&D Systems. Defined fetal bovine serum (FBS) was purchased from HyClone, vitronectin was from Thermo Fisher Scientific, and insulin, dexamethazone and a protease inhibitor cocktail were from Sigma. 3-Isobutyl-1-methylxanthine (IBMX) and pioglitazone were obtained from Wako. CAPE and Galiellalactone were from Tocris and Cayman Chemical, respectively. Antibodies for villin1 (ab3304), mucin 2 (Muc2) (ab11197 for human, ab76774 for mouse), and chromogranin A (ChgA) (ab15160) were purchased from Abcam. Anti-E-cadherin antibody (3195) was from Cell Signaling, anti-lysozyme antibody (A0099) was from Dako, and anti-perilipin1 antibody (GP29) was from Progen. Secondary antibodies for immunostaining were from Jackson ImmunoResearch.
3
Isolation and Differentiation of Adipocyte and Fibroblast Cells
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Preparation of the SVF from WAT was performed as previously described (8 (link)). In brief, WAT was minced with scissors and digested with 2 mg/mL Collagenase I (Sigma-Aldrich) in DMEM at 37°C for 1 h. DMEM plus 10% FBS was added to double the volume, floating adipocytes were removed, and the digest was filtered through a 100-μm mesh that retained vessels of the stromal-particulate fraction. The filtrate was centrifuged at 800g for 5 min, and the SVF pellet was resuspended in DMEM containing 10% FBS and cultured for 2 days.
Isolation of primary MEFs was carried out as previously described (35 (link)). 3T3-L1 cells and MEFs were cultured in growth medium (DMEM plus 10% FBS) at subconfluence. For adipocyte differentiation, cells were grown to confluence and switched into differentiation medium I (growth medium plus 0.5 μmol/L IBMX, 1 μg/μL insulin, 0.25 μmol/L dexamethazone, 2 μmol/L rosiglitazone; Sigma-Aldrich) for 2 days. The culture medium was then changed to differentiation medium II (growth medium plus 2 μmol/L rosiglitazone), and the medium was changed every 2 days. To activate SHH signaling, MEFs were treated with 250 ng/mL SHH (R&D Systems) or 5.2 μmol/L purmorphamine (Calbiochem) in differentiation media from differentiation day 2 onward.
Isolation of primary MEFs was carried out as previously described (35 (link)). 3T3-L1 cells and MEFs were cultured in growth medium (DMEM plus 10% FBS) at subconfluence. For adipocyte differentiation, cells were grown to confluence and switched into differentiation medium I (growth medium plus 0.5 μmol/L IBMX, 1 μg/μL insulin, 0.25 μmol/L dexamethazone, 2 μmol/L rosiglitazone; Sigma-Aldrich) for 2 days. The culture medium was then changed to differentiation medium II (growth medium plus 2 μmol/L rosiglitazone), and the medium was changed every 2 days. To activate SHH signaling, MEFs were treated with 250 ng/mL SHH (R&D Systems) or 5.2 μmol/L purmorphamine (Calbiochem) in differentiation media from differentiation day 2 onward.
4
Adipocyte Differentiation Molecular Protocol
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Insulin, dexamethazone, BSA, MG-132, forskolin, lactacystin, E-64d, cycloheximide, anti-Flag antibody and a protease inhibitor cocktail were purchased from Sigma-Aldrich. IBMX and pioglitazone were from Wako Chemicals. ALLN and OAs were from Nacalai Tesque. Anti-PPARγ and anti-ubiquitin antibodies were from Santa Cruz Biotech. Anti-Plin1 and anti-Plin2 antibodies were from Progen. Anti-GFP and anti-actin antibodies were from Abcam and Chemicon, respectively.
5
Osteogenic Differentiation in Bioreactor
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Prior to the experiment, the scaffolds were soaked and incubated in standard culture medium for 24 h. Next, samples were placed in bioreactor in a position, which ensured a permanent flow of the medium through the scaffolds. HBDCs were suspended in 0.5 L of culture medium (9 × 105 cells per sample) and put into the bioreactor. The cells were cultured in CO2 incubator, where the whole system was placed. After 1 week the medium was changed for a fresh medium enriched with 10 nM dexamethazone (Sigma, USA), 10 nM vitamin D3 (Sigma, USA) and 10 mM beta-glycerophosphate (Sigma, USA). From this time point 50 mL of medium was changed twice a week. The samples in bioreactor were cultured for 5 weeks.
6
Osteogenic Differentiation of Immortalized Human MSCs
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An immortalised cell line of human bone marrow mesenchymal stem cells (MSCs) [26] was seeded at density of 3x10 4 cells/ml in standard MSC medium consisting of Dulbecco's Modified Eagle's Medium (DMEM), 10% FCS, 1 mM L-glutamine, 1% non-essential amino acids and 10% antibiotics, and incubated at 37°C in 5% CO 2 . For differentiation, confluent cells were treated with osteogenic medium (OS) consisting of standard MSC medium supplemented with 100 nm dexamethazone (Sigma-Aldrich, Gillingham, UK), 0.05 mM L-ascorbic acid 2 phosphate (Acros Organics, Geel, Belgium) and 10 mM beta glycerophosphate (βGP, Sigma-Aldrich) for 21 days with medium changes every 2 days. Parallel cultures maintained in standard MSC medium were used as untreated controls (C).
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