3T3-L1 cells were obtained from Zenbio, Inc. at passage 8 (Cat. # SP-L1-F, lot # 3T3062104; Research Triangle Park, NC) and were maintained as described previously in pre-adipocyte maintenance media (Dulbecco’s Modified Eagle Medium – High Glucose (DMEM-HG; Gibco # 11995), 10% BCS, 1% penicillin and streptomycin (Pen-Strep; Gibco # 15140)) (Kassotis et al., 2017a (link); Kassotis et al., 2017b (link)). Cells were maintained in a sub-confluent state until differentiation and each thaw of frozen cells was utilized within 6 passages, with no significant changes in control chemical response observed in that time. HEK 293H cells utilized for the GeneBlazer™ PPARγ assays (Invitrogen # K1094) were cultured in DMEM-HG supplemented with 10% fetal bovine serum (FBS, Invitrogen # 26400-036), 1% non-essential amino acids (Invitrogen # 11140), 1% sodium pyruvate (Invitrogen # 11360), 1% Pen-Strep, 1 M HEPES (Invitrogen # 15630), 80 μg/ml hygromycin (Invitrogen # 10687), 1% glutamax (Invitrogen # 35050), and 500 μg/ml Geneticin (Invitrogen # 10131). Each thaw of HEK 293H cells was maintained in a sub-confluent state and utilized for assays within 10 passages.
3t3 l1 cells
Manufactured by ZenBio
Sourced in United States
3T3-L1 cells are a well-established cell line derived from mouse embryonic fibroblasts. They are commonly used as a model system for studying adipocyte (fat cell) differentiation and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Dmem hg, by Thermo Fisher Scientific (3 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
Isobutylmethylxanthine, by Merck Group (1 mentions)
Fatty acid free bovine serum albumin, by Roche (1 mentions)
8 bromo cyclic adenosine monophosphate camp, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Bt474, by American Type Culture Collection (1 mentions)
Skbr3, by American Type Culture Collection (1 mentions)
Dm 2 l1, by ZenBio (1 mentions)
Hadscs, by Lonza (1 mentions)
Dulbecco s modified eagle s medium dmem, by Nacalai Tesque (1 mentions)
10 protocols using 3t3 l1 cells
1
Adipocyte Differentiation and Gene Reporter Assay
2
Differentiation and Culture of 3T3-L1 and HEK 293H Cells
3
3T3-L1 Adipocyte Differentiation Protocol
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3T3-L1 cells were purchased from Zen-Bio (Research Triangle Park, NC, USA). Dulbecco's modified Eagle's medium and other culture reagents were purchased from Thermo Scientific (Rockford, IL, USA). Fatty acid-free bovine serum albumin was purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). Isobutyl methyl xanthine, dexamethasone, insulin, isoproterenol (ISOP) and 8-Bromo-cyclic adenosine monophosphate (cAMP) were purchased from Sigma (St Louis, MO, USA). Human HDL was obtained as a gift from Dr Jay Heinecke (Department of Medicine, University of Washington, Seattle, WA, USA), and the method of HDL preparation has been published previously.26 (link) Human apoA-I was purified as previously described.27 (link)
4
Maintenance and Differentiation of 3T3-L1 Cells
5
3T3-L1 Cell Culture and Differentiation
6
3T3-L1 Adipocyte Differentiation Protocol
7
Adipogenic Differentiation of 3T3-L1 and Ing-svf Cells
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hADSCs were purchased from Lonza. 3T3-L1 cells were purchased from ZenBio and used at low passage. Ing-svf cells were a gift from R. Gupta (24 (link)). Low-passage cells were cultured in DMEM (Corning, 10-0170CV) containing high glucose and pyruvate, supplemented with 10% FBS, 1% pen/strep at 37°C, and 10% CO2. Cells were grown to confluence. Two days after confluency, adipogenic differentiation cocktail was added for 2 days: 0.25 μM dexamethasone, insulin (1 μg/ml), 0.5 mM IBMX, and 2 μM rosiglitazone for 3T3-L1 cells and 1 μM dexamethasone, insulin (5 μg/ml), 0.5 mM IBMX, and 1 μM rosiglitazone for Ing-svf cells. Differentiated cells were then maintained in enriched DMEM with insulin (2 μg/ml) or insulin (5 μg/ml) for 3T3-L1 and Ing-svg, respectively.
8
Determining Cell Viability via MTT Assay
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Cell viability assay was performed using MTT21 (link) with modification. The 3T3-L1 cells (Zen-Bio Inc., USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque Inc., Japan) supplemented with 10% FBS and 1% antibiotics. Cells were seeded at a density of 2 × 105 cells/mL and incubated at 37°C with 5% CO2. After reaching confluence, the cells were treated with samples and further incubated for 48 h. Once the treatment period was over, MTT solution (5 mg/mL) was added to each well, and the plate was incubated at 37°C with 5% CO2 for 4 h. Next, the supernatant was removed, and 100 µL of DMSO was added to each well after which spectrophotometric absorbance was measured at a wavelength of 570 nm with a reference wavelength of 630 nm. The 50% reduction in cell number relative to the control (IC50) was established by extrapolation from a graph of the experimental data.
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Cell viability assay was performed using MTT21 (link) with modification. The 3T3-L1 cells (Zen-Bio Inc., USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque Inc., Japan) supplemented with 10% FBS and 1% antibiotics. Cells were seeded at a density of 2 × 105 cells/mL and incubated at 37°C with 5% CO2. After reaching confluence, the cells were treated with samples and further incubated for 48 h. Once the treatment period was over, MTT solution (5 mg/mL) was added to each well, and the plate was incubated at 37°C with 5% CO2 for 4 h. Next, the supernatant was removed, and 100 µL of DMSO was added to each well after which spectrophotometric absorbance was measured at a wavelength of 570 nm with a reference wavelength of 630 nm. The 50% reduction in cell number relative to the control (IC50) was established by extrapolation from a graph of the experimental data.
9
Differentiation of 3T3-L1 Fibroblasts
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3T3-L1 cells were obtained from Zen-Bio (Durham, NC). 3T3-L1 cells stably transfected with pBabe-myc7-Glut4 were described previously15 (link). 3T3-L1 fibroblasts were grown in DMEM containing 10% CBS. Three days after confluence, cells were transferred to the differentiation medium (DMEM with 10% FBS, 0.174 μM insulin, 1 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine). Three days later, differentiation medium was replaced with DMEM containing 10% FBS for 4 more days until over 90% of differentiation was achieved.
10
3T3-L1 Cell Differentiation Protocol
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3T3-L1 cells were obtained from Zen-Bio (Durham, NC). 3T3-L1 cells stably transfected with pBabe-myc7-Glut4 were described previously 16 . 3T3-L1 fibroblasts were grown in DMEM containing 10% CBS. Three days after confluence, cells were transferred to the differentiation medium (DMEM with 10% FBS, 0.174 μM insulin, 1 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine). Three days later, differentiation medium was replaced with DMEM containing 10% FBS for 4 more days until over 90% of differentiation was achieved.
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