A total of 25 ml of EOLG-DM is prepared mixing 15 ml of DMEM/F12 with 0.5 ml of B27, 0.25 ml of N2, 0.25 ml of GlutaMax (2 mM), 0.25 ml of Penicillin-streptomycin (50 U/ml), 0.25 ml of Non-essential amino acid solution (0.1 mM), 0.25 ml of sodium pyruvate (1 mM; all purchased from Thermo Fisher Scientific), 50 μl of triiodo-l-thyronine (T3, 40 ng/ml, stock concentration: 20 μg/ml; Sigma-Aldrich), 20 μl of sonic hedgehog (Shh) (40 ng/ml stock, concentration: 50 μg/ml), 20 μl of Noggin (40 ng/ml, stock concentration: 50 μg/ml), 5 μl of insulin-like growth factor (IGF, 100 ng/ml, stock concentration: 500 μg/ml; all purchased from PeproTech), and 25 μl of neurotrophin 3 (NT-3, 10 ng/ml; Sigma-Aldrich). DMEM/F12 is added to a final volume of 25 ml, filter with a bottle-top filter (0.22 μm) and store at 4°C. We suggest to use complete media within 1 week.
Triiodo l thyronine t3
Manufactured by Merck Group
Sourced in Germany, Australia
Triiodo-l-thyronine (T3) is a lab equipment product manufactured by Merck Group. It is a thyroid hormone commonly used in research and diagnostic applications. T3 is a key regulator of cellular metabolism and plays a crucial role in various physiological processes.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Insulin, by Merck Group (3 mentions)
Indomethacin, by Merck Group (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
L thyroxine t4, by Merck Group (2 mentions)
Nonessential amino acid solution, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Noggin, by Thermo Fisher Scientific (1 mentions)
Insulin like growth factor igf, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Rosiglitazone, by Merck Group (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
Fm19g11, by Merck Group (1 mentions)
Ms 222, by Merck Group (1 mentions)
Ficoll, by Merck Group (1 mentions)
Human chorionic gonadotropin (hcg), by Aska Pharmaceutical (1 mentions)
3 isobutyl 1 methylxanthine ibmx, by Merck Group (1 mentions)
Atl buffer, by Qiagen (1 mentions)
10 protocols using triiodo l thyronine t3
1
Optimized EOLG-DM Culture Media
2
Isolation and Differentiation of Adipose Stromal Cells
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The primary stromal vascular fraction from iBAT of newborn ICR mice was obtained by collagenase digestion. The digested iBAT was filtered through a 100-μm nylon cell strainer, and the cells were isolated by centrifugation (156g) for 5 min. The cell pellet was washed with PBS and cultured with DMEM supplemented with 20% FBS. The cells were seeded, grown to confluence (designated day 2) and cultured with differentiation induction medium (DMEM [Sigma-Aldrich] supplemented with 20% FBS, 20 nM insulin [Sigma-Aldrich], 1 nM triiodo-L-thyronine [T3; Sigma-Aldrich], 5 μM dexamethasone, 0.125 mM indomethacin [Sigma-Aldrich], 0.5 mM IBMX [Sigma-Aldrich], and 1 μM rosiglitazone [Sigma-Aldrich]) on day 0. After differentiation induction, the cells were cultured with differentiation enhancement medium (DMEM supplemented with 20% FBS, 20 nM insulin, and 1 nM T3) on days 2 and 4. HEK293 cells were maintained in DMEM containing 10% FBS and penicillin–streptomycin (glycolysis conditions). For habituation to OXPHOS conditions, HEK293 cells were cultured with glucose-free DMEM (Nacalai tesque) containing 10% FBS, 1 mM sodium pyruvate, 10 mM galactose and penicillin–streptomycin and incubated for 12 or 24 h.
3
Inhibition of HIF-1α by FM19G11 in Cells
4
Xenopus tropicalis Embryonic Development
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Xenopus tropicalis, the Golden strain, were provided by the Amphibian Research Center (Hiroshima University) through the National Bio-Resource Project of the AMED, Japan. Eggs and testes were collected from sexually mature adult frogs with an injection of human chorionic gonadotropin (Aska Pharmaceutical, Tokyo, Japan), and in vitro fertilization was carried out. To isolate testes, male frogs were treated with 1% MS-222 (tricaine; Sigma-Aldrich, MO, USA) as anesthesia and euthanasia. One-cell-stage embryos were de-jellied with 2% L-cysteine solution. After washing with 0.1×Marc's modified ringer (MMR), embryos were microinjected in 6% Ficoll (Sigma-Aldrich) in 0.33×MMR. At the blastula stage, embryos were moved to 0.1×MMR. At 16–20 h after fertilization, normally developed embryos were collected and counted and represented the initial numbers of individuals in each experiment. Embryos and tadpoles were reared at 25–26°C. Tadpoles at stage 52–54 were treated with 10 nM 3,3,5-triiodo-L-thyronine (T3; Sigma-Aldrich) for 3 days. Animal rearing and treatments were performed and approved according to the Hiroshima University guidelines for the use and care of experimental animals.
5
Oligodendrocyte Differentiation Protocol
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To induce differentiation into mature oligodendrocytes, the OPCs were seeded into a 24-well plate at a concentration of 5×104 cells/ml per well. After 24 h the cells were cultured in oligodendrocyte differentiation medium [OLM; DMEM/F12 supplemented with 2% B27 (Invitrogen; Thermo Fisher Scientific, Inc.), Triiodo-L-thyronine (T3; 40 ng/ml) and L-Thyroxine (T4; 30 ng/ml) (both from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)] for 5 days with the medium changed every day.
6
Mouse Preadipocyte Differentiation Protocol
7
Monocyte Activation Response to T3 and LPS
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Isolated monocytes were seeded in 6-well plates (9.6 cm2/well) at a density of 2 × 106 monocytes/well in 2 mL RPMI culture medium for 1 h at 37 °C, with visual confirmation of attachment by light microscopy. The seeding medium was then aspirated from the plate and the attached monocytes were treated with either RPMI medium, 5 µM triiodo-L-thyronine (T3) (Sigma, Victoria, Australia, T0281), 10 ng/mL lipopolysaccharide (LPS) (E. coli O55:B5, Sigma, Victoria, Australia, L2880), or a costimulation of 5 µM T3 and 10 ng/mL LPS (T3 + LPS) (with monocytes pre-exposed to T3 for 1 h prior to the addition of LPS) for 4 h or 24 h at 37 °C. All treatments were administered at a volume of 2 mL per well, with RPMI used as the diluent. Separate wells were used for the subsequent RNA and DNA collection. Monocytes for subsequent RNA extraction were collected in 500 µL Trizol and stored at −80 °C. Monocytes for subsequent DNA extraction were collected in 500 µL of ATL buffer (QIAGEN, Victoria, Australia, 56304) and stored at −30 °C. In addition to 4 h and 24 h treatments, untreated monocytes were also collected for DNA and RNA extraction following seeding as a time zero (0 h) control.
8
BEM + EPO and T3 Supplementation
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BEM supplemented with 2U/ml EPO, 1µM Triiodo-L-Thyronine (T3, SIGMA)
9
Adipocyte Differentiation Protocol
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Mouse preadipocytes, 293T cells and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% Fetal Bovine Essence (FBE; VWR, #10803-034) and penicillin/streptomycin (Thermo Fisher Scientific, #15140122). CAD cells were cultured in DMEM/nutrient mixture F-12 (DMEM/F12) (Thermo Fisher Scientific, #11320033) supplemented with 8% fetal bovine serum (FBS; Hyclone, # SH30088.03HI). All cell lines were maintained in a humidified incubator at 37°C with 5% CO2.
To differentiate preadipocytes into mature adipocytes, preadipocytes were grown to ~95% confluence before a differentiation cocktail was added at the following final concentrations: insulin (5 μg/ml; Sigma-Aldrich, #I0516), 1 nM triiodo-l -thyronine (T3; Sigma-Aldrich, #T2877), 125 μM indomethacin (Sigma-Aldrich, #I-7378), 5 μM dexamethasone (Sigma-Aldrich, #D1756), and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, #I5879). After 2 days, the cells were switched to DMEM supplemented with 10% FBE, insulin (5 μg/ml), and 1 nM T3. After another 2 days, fresh DMEM media supplemented with 10% FBE and 1 nM T3 were added to the cells. Differentiated adipocytes were usually analyzed 6 days after addition of the differentiation cocktail.
To differentiate preadipocytes into mature adipocytes, preadipocytes were grown to ~95% confluence before a differentiation cocktail was added at the following final concentrations: insulin (5 μg/ml; Sigma-Aldrich, #I0516), 1 nM triiodo-
10
Isolation and Differentiation of Oligodendrocyte Progenitor Cells
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OPCs were prepared from whole brains of newborn (P1-P3) ADAMTS4 -/-KO or C57Bl/6J wild-type mice. Meninges were removed, brains were disintegrated with a scalpel, and pooled from animals with the same genotype. Brain tissue was further dissociated with the Neural Tissue Dissociation Kit, and A2B5 + glial-restricted progenitors were isolated by labeling with anti-A2B5 microbeads and magnetic enrichment using MS columns and a MiniMACS Separator according to the manufacturer's instructions (Miltenyi Biotec). 1.5 × 10 6 progenitor cells were seeded on poly-L-lysine coated 10 cm dishes and cultured in proliferation medium (DMEM/F12 without glutamine, 1× B-27 supplement without vitamin A, 1× GlutaMAX, 100 U/ml penicillin/streptomycin, 20 ng/ml each of mouse FGF-b and mouse PDGF-AA, all media components from ThermoFisher Scientific, growth factors from ImmunoTools). Confluency was kept below 50% and cells were passaged approximately every 4 days after dissociation of the cell monolayer with accutase (Sigma-Aldrich). For differentiation of OPCs, cells were switched to differentiation medium (same media components, except 1× B-27 with vitamin A, and without growth factors but instead supplemented with 40 ng/ml each of L-thyroxine (T 4 ) and triiodo-L-thyronine (T 3 ), Sigma-Aldrich) for 1-5 days.
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