The ASC fraction was isolated from fat attached to skin as described previously and grown in fibroblast medium [20 (link)]. Five days before construction of the adipose layer, adipocyte differentiation was induced by ASC differentiation medium [22 (link)] consisting of DMEM:Ham’s F12 1:1, supplemented with 1% penicillin/streptomycin, 33 µM biotin (Sigma-Aldrich), 17 µM d -pantothenic acid hemicalcium salt (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich), 100 nM humulin R (Lilly, Indianapolis, IN), 1 µM rosiglitazone (Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 2 nM 3,3′,5-triiodo-l -thyronine sodium salt (Sigma-Aldrich) and 10 µg/ml human transferrin (Sigma-Aldrich). Three days after differentiation induction, medium was changed to ASC maintenance medium (ASC-MM) consisting of DMEM:Ham’s F12 1:1, supplemented with 1% penicillin/streptomycin, 33 µM biotin, 17 µM d-pantothenic acid hemicalcium salt, 10 nM dexamethasone and 10 nM humulin R. Cells were cultured at 37 °C, 5% CO2.
3 3 5 triiodo l thyronine sodium salt
Manufactured by Merck Group
Sourced in United States
3,3',5-triiodo-l-thyronine sodium salt is a compound used in laboratory research applications. It is the sodium salt form of the thyroid hormone triiodothyronine (T3). This product is typically used as a reference standard or research tool in various biochemical and cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
3 isobutyl 1 methylxanthine, by Merck Group (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Polybrene, by Merck Group (2 mentions)
D biotin, by Merck Group (2 mentions)
Pantothenate, by Merck Group (2 mentions)
Rosiglitazone, by Enzo Life Sciences (2 mentions)
Human transferrin, by Merck Group (1 mentions)
Rosiglitazone, by Merck Group (1 mentions)
D pantothenic acid hemicalcium salt, by Merck Group (1 mentions)
Humulin r, by Eli Lilly (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
Brdu cell proliferation elisa, by Roche (1 mentions)
Saponin, by Merck Group (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti α actin, by Merck Group (1 mentions)
Bodipy phallacidin, by Thermo Fisher Scientific (1 mentions)
10 protocols using 3 3 5 triiodo l thyronine sodium salt
1
Adipocyte Differentiation from ASCs
2
Cardiac Muscle Cell Proliferation Analysis
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Chemicals used: Bodipy phallacidin (Molecular Probes, Eugene, OR); Dulbecco's Modified Eagle Medium, nonessential amino acids, sodium pyruvate, penicillin and streptomycin (PEST), fetal bovine serum (FBS), and Trypsin‐EDTA (Gibco, Paisley, Scotland); TRI‐reagent, monoclonal anti‐α‐actin antibody, insulin‐like growth factor‐1 (IGF‐1), 3, 3',5‐Triiodo‐L‐thyronine sodium salt (T3), anti‐α‐actin, bovine serum albumin (BSA), protease, and saponin (Sigma Chemical Co., St. Louis, MO); CellTiter 96® Aqueous One solution cell proliferation (MTS) assay (Promega, Madison, WI); paraformaldehyde (PFA) (Labkemi, Stockholm, Sweden); BrdU Cell Proliferation ELISA (Roche Diagnostics Corporation, Indianapolis, IN); monoclonal α‐actinin antibody (Abcam, Cambridge, UK); polyclonal Ddr‐2 antibody (Santa Cruz, Santa Cruz, CA); Prolong gold antifade reagent (Invitrogen, Carlsbad, CA); Collagenase type 2 (Worthington, Lakewood, NJ).
3
Differentiation of Stem Cells to Thyroid
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Cells were further cultured in MCDB 131 medium supplemented with 1.5 g/l sodium bicarbonate, 1 × Glutamax, 20 mM glucose, 2% BSA, 0.25 μM SANT-1, 0.05 μM retinoic acid, 100 nM LDN193189, 1:200 ITS-X, 1 μM T3 (3,3′,5-Triiodo-l -thyronine sodium salt, Sigma, T6397), 10 μM ALK5 inhibitor II (Enzo Life Sciences, NY, Cat #ALX-270-445), 10 μM zinc sulfate (Sigma, Z0251) and 10 μg/ml of heparin (Sigma, H3149).
4
Lentiviral Infection and Adipogenic Differentiation of Human Myoblasts
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Human myoblasts were grown in the incomplete SkBM medium until 90-100% confluence in 12-well plates and were infected by exposure to lentiviral supernatant (∼ 4 MOI) and polybrene (0.8 μg/mL, Millipore, Billerica, MA) daily for two days. After the second lentiviral infection, human myoblasts or AdSVCs underwent adipogenesis according to the non-modified adipogenesis protocol (23 (link)). Briefly, the cells were cultured in the adipocyte induction medium DMEM/F12 (Gibco) containing d-Biotin (33 nM, Sigma), human insulin (70 nM, Sigma I9278), dexamethasone (100 nM, APP pharmaceuticals, Schaumburg, IL), pantothenate (4 μg/mL, Sigma), human transferrin (10 μg/mL, Calbiochem), 3,3′, 5-triiodo-L-thyronine sodium salt (2 μM, Sigma), rosiglitazone (1μM, Enzo Lifesciences, Farmingdale, NY), isobutylmethylxanthine (IBMX, 0.5 mM, Sigma), 10% FBS, 100U/mL penicillin-streptomycin. After 72 hr, cells were switched to the adipocyte differentiation medium (induction medium without rosiglitazone and IBMX) for additional 8 days or until collection.
5
Differentiation of Rat MG Epithelial Cells
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For ALI culture, passage 2 rat MG epithelial cells were seeded onto collagen precoated 12 mm permeable Snapwell inserts (Corning-Costar, Cambridge, MA, USA) at a density of 120 k/well in F medium.20 (link) On the following day, cells were fed with differentiation medium (DMEM/F-12 1:1 [vol/vol; Invitrogen Life Technologies], supplemented with 2% Ultroser G [Pall Corporation, Port Washington, NY, USA], 2% HyClone FetalClone II Serum [GE Healthcare Bio-sciences, Pittsburgh, PA, USA], 22.5 μg/mL bovine brain extract [Lonza, Hopkinton, MA, USA], 2.5 μg/mL transferrin [Invitrogen Life Technologies], 2.5 μg/mL insulin [Sigma-Aldrich Corp.], 20 nM hydrocortisone [Sigma-Aldrich Corp.], 500 nM 3,3′,5-Triiodo-L-thyronine sodium salt [Sigma-Aldrich Corp.], 1.5 μM epinephrine [Sigma-Aldrich Corp.], 10 nM retinoic acid [Sigma-Aldrich Corp.], 250 nM O-Phosphorylethanolamine [Sigma-Aldrich Corp.], 250 nM ethanolamine [Sigma-Aldrich Corp.], 100 U/mL penicillin [Sigma-Aldrich Corp.], and 100 μg/mL streptomycin [Sigma-Aldrich Corp.]). Three days after seeding, the apical medium was removed to create ALI cultures, which were maintained for one additional week.
6
Thyroid Hormone Effects on HepG2 Cells
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HepG2 cells (#ACC 180; DSMZ, Braunschweig, Germany) were cultured in RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 μg/ml of streptomycin (all from Gibco, Thermo Fisher Scientific, Karlsruhe, Germany). For the experiments, 350,000 cells were seeded per well in a 12-well plate, grown for 24 h, and then treated with 10 nM 3,3′,5-triiodo-l -thyronine sodium salt (Sigma–Aldrich, Schnelldorf, Germany) for another 24 h. Control cells were incubated with the appropriate volume of vehicle (NaOH).
7
Differentiation of Murine Brown Pre-adipocytes
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The immortalized murine-derived brown pre-adipocyte cell line (IBA) was derived from the stromal vascular fraction (SVF) of interscapular brown adipose tissue of young male (C57BL6/J)20 provided by Prof. Christian Wolfrum’s laboratory (ETH-Zurich) and prepared according to standard protocols. IBA was cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% fetal calf serum (FCS, AMIMED), with 100X penicillin/streptomycin/glutamine (100x Pen/Strep/Glutamine, Life Technologies) in a 5% CO2 humidified atmosphere at 37 °C and maintained at less than 80% confluence before passaging. Differentiation of IBAs was induced by exposing confluent cells (Day 0) to DMEM containing 10% FCS supplemented with 0.5 mM 3-isobutyl-1-methylxanthine, 1 uM dexamethasone, 1.96 nM insulin (Sigma, stock solution of 167uM prepared in PBS), 125 uM indomethacin (Sigma, stock solution of 18 mM prepared in absolute ethanol), 1 nM T3 (3,3′,5-Triiodo-L-thyronine sodium salt, Sigma, prepared in 45 mM KOH). After exactly 28 hours, the induction medium was replaced with adipocyte differentiation medium consisting of 1 nM T3 and 1.96 nM insulin. Cells were maintained in this medium until Day 5 and were stained with Oil Red O for lipid accumulation at Day 6 as full differentiation is achieved by then.
8
T3 Treatment Effects on HepG2 Cells
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HepG2 cell line was purchased from the Bioresource Collection and Research Center (Food Industry Research and Development Institute, Hsinchu, Taiwan). Cells were maintained (5% CO2, 37 °C) in Dulbecco’s modified eagle medium (HyClone, South Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum and antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin). After an overnight starvation from fetal bovine serum, the cells were treated with 3,3′,5-triiodo-L-thyronine sodium salt (Sigma-Aldrich, St. Louis, MO, USA) as T3 treatment with variable concentration and durations.
9
Lentiviral Infection and Adipogenic Differentiation of Human Myoblasts
10
Oligodendrocyte Terminal Differentiation
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For terminal differentiation, six-well plates were coated with a substrate of 50 mg/ml poly-l-ornithine hydrobromide (Sigma-Aldrich) and 20 mg/ml laminin (Life Technologies). The plates were first coated with 1 ml poly-l-ornithine hydrobromide/well and incubated at room temperature for 1 h.
Next, the cells were coated with 1 ml laminin/well and incubated at room temperature for 1 h. After passage, cells were then cultured in OPC expansion medium for 1 week.
Confluent OPCs were passaged at a density of 1,000 cells per well and cultured in OPC expansion medium for 1 week, with medium changes every other day. The cells were then cultured in oligodendrocyte terminal differentiation medium [adapted from Wang et al. (41) ], with medium changes every other day. This medium consisted of DMEM/F12 without phenol red or HEPES, 0.25× N1 medium supplement (Sigma-Aldrich), 0.5× N-2 supplement, 1.25× B-27 supplement, serum free, 30 ng/ml 3,3¢,5-triiodo-l-thyronine sodium salt (Sigma-Aldrich), 50 ng/ml biotin (Sigma-Aldrich), 0.5 mM dibutyryl cAMP (Sigma-Aldrich), 2.5 ng/ml recombinant human plateletderived growth factor (PDGF-AA) (Peprotech, Rocky Hill, NJ, USA), 5 ng recombinant human BDNF (Peprotech), 2.5 ng/ml recombinant human insulin-like growth factor-1 (IGF-1) (Peprotech), 2.5 ng/ml NT3 (Peprotech), 0.5× penicillin/streptomycin, and 27.5 mM 2-mercaptoethanol.
Next, the cells were coated with 1 ml laminin/well and incubated at room temperature for 1 h. After passage, cells were then cultured in OPC expansion medium for 1 week.
Confluent OPCs were passaged at a density of 1,000 cells per well and cultured in OPC expansion medium for 1 week, with medium changes every other day. The cells were then cultured in oligodendrocyte terminal differentiation medium [adapted from Wang et al. (41) ], with medium changes every other day. This medium consisted of DMEM/F12 without phenol red or HEPES, 0.25× N1 medium supplement (Sigma-Aldrich), 0.5× N-2 supplement, 1.25× B-27 supplement, serum free, 30 ng/ml 3,3¢,5-triiodo-l-thyronine sodium salt (Sigma-Aldrich), 50 ng/ml biotin (Sigma-Aldrich), 0.5 mM dibutyryl cAMP (Sigma-Aldrich), 2.5 ng/ml recombinant human plateletderived growth factor (PDGF-AA) (Peprotech, Rocky Hill, NJ, USA), 5 ng recombinant human BDNF (Peprotech), 2.5 ng/ml recombinant human insulin-like growth factor-1 (IGF-1) (Peprotech), 2.5 ng/ml NT3 (Peprotech), 0.5× penicillin/streptomycin, and 27.5 mM 2-mercaptoethanol.
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