3T3-L1 preadipocytes (CL-173™, ATCC, Manassas, VA, USA) were cultured in Dulbecco's modified eagle medium (DMEM) medium (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Gibco Co., Carlsbad, CA, USA) and maintained at 37°C with 5% CO2. To induce differentiation, 3T3-L1 cells were treated with methylisobutylxanthine, dexamethasone, and insulin (MDI) induction medium (1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) and 10 μg/mL insulin in 10% DMEM medium) at approximately 100% confluence. The adipocytes were allowed to differentiate for 8 days in culture. The accumulation of lipid droplets was examined.
Cl 173
Manufactured by American Type Culture Collection
Sourced in United States
The CL-173 is a laboratory centrifuge designed for general-purpose applications. It features a maximum speed of 6,000 rpm and a maximum RCF of 3,740 x g. The centrifuge can accommodate a variety of sample tubes and microplates. The core function of the CL-173 is to separate components of a liquid mixture based on their different densities.
Automatically generated - may contain errors
Lab products found in correlation
Insulin, by Merck Group (2 mentions)
Amphotericin b, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Tib 71, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Dmem medium, by Merck Group (1 mentions)
Crl 1740, by American Type Culture Collection (1 mentions)
Jurkat cell, by American Type Culture Collection (1 mentions)
X rad 320, by Precision X-Ray (1 mentions)
Ccl 226, by American Type Culture Collection (1 mentions)
Bovine calf serum (bcs), by American Type Culture Collection (1 mentions)
Viafect transfection reagent, by Promega (1 mentions)
Pt 5020, by Lonza (1 mentions)
Pt 5005, by Lonza (1 mentions)
Tib 67, by American Type Culture Collection (1 mentions)
Mycoalert, by Lonza (1 mentions)
Methylisobutylxanthine, by Merck Group (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by American Type Culture Collection (1 mentions)
33 protocols using cl 173
1
3T3-L1 Preadipocyte Differentiation Protocol
2
Culturing Murine and Human Prostate Cancer Cell Lines
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Murine embryonic fibroblast 3T3-L1 cells (ATCC, #CL-173™) were maintained in a proliferative medium (PM) made up of DMEM high glucose (4.5 g/L glucose), supplemented with 10% newborn calf serum, 4 mM L-glutamine, 1 mM, sodium pyruvate and 1% antibiotic–antimycotic cocktail (100 U/mL penicillin, 10 µg/mL streptomycin, 0.25 µg/mL amphotericin B). Murine PCa cells TRAMP-C1 (ATCC, #CRL-2730™) were cultured in DMEM high glucose supplemented with 5% fetal bovine serum (FBS), 4 mM L-glutamine, 5% Nu-serum IV complement, 0.005 mg/mL insulin, 10 nM dehydroepiandrosterone (DHEA) and 1% of an antibiotic–antimycotic cocktail. Human androgen-dependent LNCaP cells (ATCC, #CRL-1740) were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 15 mM HEPES, and 1% of antibiotic cocktail (100 U/mL penicillin, 10 µg/mL streptomycin). Human androgen-independent PC-3 cells (ATCC, #CRL-1435) were cultured in DMEM/F12 medium supplemented with 10% FBS, 2 mM L-glutamine, and 1% of an antibiotic–antimycotic cocktail. All cell lines were cultured at standard conditions (Tª: 37 oC, CO2: 5%). FBS was stripped from small weight molecules, including steroids, by using a protocol previously published [16 (link)]. Charcoal-stripped FBS (csFBS) was employed to perform a cell culture medium without androgens as a substitute of FBS.
3
Radiation Effects on Immune Cells
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Jurkat cells (Clone E6–1, ATCC, VA, USA) were irradiated with doses of 0, 1, 3, or 6Gy (Xrad 320, Precision X-ray Inc., CT, USA) and left to recover for 24 h. At days 1, 14, and 28, post-irradiation cells were mitogen-stimulated and analyzed by flow cytometry as described above. Adipocytes (3 T3-L1, CL-173, ATCC) were irradiated with doses of 0, 1, 3, and 6Gy and left to recover for 24 h prior to the collection of culture media. PBMCs from six young healthy donors were mitogen-stimulated in conditioned media from irradiated adipocytes and analyzed by flow cytometry as described above.
4
Differentiation of 3T3-L1 Preadipocytes
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3T3-L1 preadipocytes were obtained from ATCC (Catalogue # CL-173™) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics (100 U mL−1 of penicillin G and 100 μg mL−1 of streptomycin) at 37 °C in a humidified atmosphere containing 5% CO2. To obtain fully differentiated adipocytes, 3T3-L1 preadipocytes were grown into 2-day post-confluence, and then differentiation was induced by using a standard differentiation cocktail containing 0.5 mM of IBMX, 1 μM of dexamethasone, 1 μg mL−1 of insulin, and 10% FBS. After 48 h, medium was changed into DMEM supplemented with 10% FBS and 1 μg mL−1 of insulin and replenished every 48 h for an additional 6 days.
5
Adipogenic Differentiation of C3H10T1/2 and 3T3-L1 Cells
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C3H10T1/2 cells (Clone 8, ATCC® CCL‐226™) were propagated in DMEM containing 10% FCS and 1% penicillin/streptomycin until confluency. Adipogenic differentiation was induced by medium supplemented with 0.25 μM dexamethasone, 0.5 mM IBMX, 1 μg/ml insulin, and 3 nM T3 for 4 days, with a medium change every 2 days. Induction was followed by differentiation maturation medium (culture medium with 1 μg/ml insulin and 3 nM T3) for 6 days. 1 μM rosiglitazone or the corresponding concentration of DMSO was added to the medium throughout the differentiation. 3T3‐L1 preadipocytes (ATCC® CL‐173™) were cultured in low (1 g/l)‐glucose DMEM containing 10% FCS and 1% penicillin/streptomycin until confluency. Adipogenic differentiation was induced with high (4.5 g/l)‐glucose DMEM containing 10% FCS and 1% penicillin/streptomycin supplemented with 0.5 μM dexamethasone, 0.5 mM IBMX, 1 μg/ml insulin for 4 days. Following induction, cells were cultured in high (4.5 g/l)‐glucose DMEM with 10% FCS, 1% penicillin/streptomycin, 1 μg/ml insulin for an additional 6 days and further 2 days without insulin. 1 μM rosiglitazone or the corresponding concentration of DMSO was added to the medium up to day 6.
6
Knockdown of Thm1 in 3T3-L1 Preadipocytes
7
Cellular Model Maintenance and Characterization
8
Evaluation of Swertiamarin's Anti-inflammatory and Anti-adipogenic Effects
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The RAW264.7 (TIB-71; ATCC, Manassas, VA, USA) murine monocytic cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% foetal bovine serum (FBS; Gibco) until the cells reached 90% confluence. Then, the cells were serum-starved for 6 h and incubated with 10 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) in the presence of swertiamarin for 16 h. The LPS-induced inflammatory signals were examined by western blotting.
3T3-L1 mouse pre-adipocytes (CL-173, ATCC) were cultured in DMEM supplemented with 10% FBS until confluent. At 2 days post-confluency, the medium was replaced with DMEM containing 0.5 mM methylisobutylxanthine (Sigma-Aldrich), 0.125 mM indomethacin (Sigma-Aldrich), and 1.0 µg/mL insulin (Sigma-Aldrich). After 2 days, the induced cells were cultured in maintenance medium (DMEM containing 10% FBS, 1.0 µg/mL insulin, and 1 nmol/L T3) for 5 days and treated with swertiamarin at the indicated concentrations for 48 h. Oil Red O staining and quantitative real-time polymerase chain reaction (qPCR) were performed to evaluate the lipid content of cells.
3T3-L1 mouse pre-adipocytes (CL-173, ATCC) were cultured in DMEM supplemented with 10% FBS until confluent. At 2 days post-confluency, the medium was replaced with DMEM containing 0.5 mM methylisobutylxanthine (Sigma-Aldrich), 0.125 mM indomethacin (Sigma-Aldrich), and 1.0 µg/mL insulin (Sigma-Aldrich). After 2 days, the induced cells were cultured in maintenance medium (DMEM containing 10% FBS, 1.0 µg/mL insulin, and 1 nmol/L T3) for 5 days and treated with swertiamarin at the indicated concentrations for 48 h. Oil Red O staining and quantitative real-time polymerase chain reaction (qPCR) were performed to evaluate the lipid content of cells.
9
Maintenance and Differentiation of iPSC-CMs
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iPSC-CM were commercially obtained directly from Axiogenesis [note this company has recently merged with Pluriomics to form NCardia] (#Ax-B-HC02-1M) and Cellular Dynamics International (CDI; #CMC-100-012-000.5). Upon receipt, cells were transferred to liquid nitrogen for long term storage. Thereafter, when required for use, cells were retrieved and plated according to manufacturer’s instructions into wells of a 96-well plate coated with fibronectin from bovine plasma (Sigma) at a final concentration of ~2 μg/cm2. Axiogenesis and CDI iPSC-CM were plated as recommended at a density of 35,000 or 50,000 viable cells per well respectively, unless stated otherwise. Cells were maintained in a sterile humidified incubator (37°C, 5% CO2) in the appropriate maintenance medium provided by the manufacturers, and was replaced 4–24 hours post plating, and every 24–48 hours thereafter.
3T3-L1 fibroblasts were obtained directly from ATCC (#CL-173) and grown and differentiated as outlined previously [25 (link)].
3T3-L1 fibroblasts were obtained directly from ATCC (#CL-173) and grown and differentiated as outlined previously [25 (link)].
10
3T3-L1 Adipocyte Differentiation Protocol
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3T3-L1 cells (CL-173, ATCC) were cultured in DMEM with L-glutamine and sodium pyruvate (high glucose, 08458-45, Nacalai tesque), supplemented with 2-mercaptoethanol (198-15781, Wako), 10% fetal bovine serum bio-equivalent (EquaFETAL, EF-0500-A, Atlas), 100 units mL−1 penicillin and 100 µg mL-1 streptomycin (168-23191, Wako) at 37 °C, and 5% CO2. The cells were cultured on culture dishes (100 mm in diameter, 150464, Thermo Fisher) and subcultured by 10-fold dilution 2–3 times per week before cultures become 60% confluent. The differentiation of 3T3-L1 cells into adipocyte-like cells was based on the method reported by Rubin et al58 (link). Briefly, 3 × 105 cells in 10 mL of culture medium were seeded on the culture dishes (100 mm in diameter) 4 days prior to stimulation for the differentiation. To start the differentiation, cell culture supernatant was replaced with a fresh culture medium containing 0.5 mM 3-isobutyl-1-methylxanthine (I5879, Sigma–Aldrich), 0.25 µM dexamethasone (D4902, Sigma–Aldrich), and 10 µg mL−1 insulin (097-06474, Wako). After 2 days, the culture supernatant was replaced with a fresh culture medium containing 1 µg mL−1 insulin. Cells on the 5th day after starting the differentiation were used for experiments. The culture medium was replaced with appropriate fresh medium every two days.
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