Mouse 3T3-L1 adipocytes (American Type Culture Collection (ATCC), Manassas, VA) were grown and maintained at no higher than 70% confluence in Dulbecco’s Modified Eagle Medium (Gibco, Camarillo, CA) supplemented with 10% fetal bovine serum (Cellgro, Manassas, VA), penicillin and streptomycin (growth medium) at 37°C in a 10% CO2 incubator. Medium was replaced every other day until cells reached confluence. For differentiation into mature 3T3-L1 adipocytes, cells were grown 2-day post confluence in growth medium and then induced to differentiate in growth medium supplemented with insulin, 3-isobutyl-1-methylxanthine and dexamethasone (Cayman Chemical, Ann Arbor, MI, USA) as described previously [18 (link)]. Three days post induction, medium was replaced with insulin only medium (growth medium supplemented with only insulin) for additional five to six days. Media was replaced every other day during this period and accumulation of lipid droplet was monitored under microscope. At least 95% of the cells showed an adipocyte phenotype at the end of differentiation period. For EV generation, differentiated 3T3-L1 adipocytes were treated for 24 h in the absence or presence of palmitic acid complexed with 1% bovine serum albumin (BSA, free fatty acid free, endotoxin free) (Sigma Chemical Co., St. Louis, MO, USA).
3 isobutyl 1 methylxanthine
Manufactured by Cayman Chemical
Sourced in United States
3-isobutyl-1-methylxanthine is a chemical compound used in research and development. It functions as a phosphodiesterase inhibitor. No further details on intended use are provided.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Cayman Chemical (5 mentions)
Insulin, by Cayman Chemical (3 mentions)
Rolipram, by Cayman Chemical (3 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Dulbecco s modified eagle s medium dmem, by Corning (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Indomethacin, by Merck Group (2 mentions)
Ly294002, by Merck Group (2 mentions)
Pki 14 22 amide, by Bio-Techne (2 mentions)
Milrinone, by Selleck Chemicals (2 mentions)
Water soluble dexamethasone dex, by Merck Group (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
3t3 l1 mouse adipocytes, by American Type Culture Collection (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Hla dr, by BD (1 mentions)
Hepes, by AppliChem (1 mentions)
Cd105, by BD (1 mentions)
Indomethacin, by Cayman Chemical (1 mentions)
Sodium pyruvate, by AppliChem (1 mentions)
13 protocols using 3 isobutyl 1 methylxanthine
1
Differentiation and EV Generation in 3T3-L1 Adipocytes
2
Multilineage Differentiation of MSCs
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50,000 cells were labeled with 3 µl antibody or corresponding isotype control (Isotype IgG2a (BD, #553456), CD45 (BD, #555492), HLA-DR (BD, #559866), CD14 (BD, #557153), CD73 (BD, #561254), CD105 (BD, #561443), Isotype IgG2aκ (BD #555573), Isotype IgG1 (Milteny, #130-081-002), CD11b (Milteny, #130-081-201), CD34 (Milteny, #130-092-213), CD90 (Milteny, #130-095-403), CD19 (Milteny, #130-091-328)) and analyzed using a Beckton Dickinson FACS Calibur).
To induce adipogenesis, confluent monolayers were treated with DMEM containing 1 μM dexamethasone, 0.01 mg/ml insulin (Berlinchemie), 0.2 mM indomethacin (Cayman Chemical Company), and 0.5 mM 3-isobutyl-1-methyl-xanthine (Serva) for 4 weeks. After fixation with 4% paraformaldehyde, staining was performed with 6 ml of 0.5% Oil red O (Sigma) in isopropanol added to 4 ml deionized water.
Chondroblastic differentiation was tested in pellet cultures with 1 × 106 cells in 4 ml DMEM containing 1 mM sodium pyruvate (Applichem), 20 mM HEPES (Roth) pH 7.3, 0.1 µM dexamethasone, 0.1 mM 2-phospho-L-ascorbic acid, and 10 ng/ml TGF-β1 (R&D). After 4 weeks, protein extraction and western blot analysis were performed.
To induce adipogenesis, confluent monolayers were treated with DMEM containing 1 μM dexamethasone, 0.01 mg/ml insulin (Berlinchemie), 0.2 mM indomethacin (Cayman Chemical Company), and 0.5 mM 3-isobutyl-1-methyl-xanthine (Serva) for 4 weeks. After fixation with 4% paraformaldehyde, staining was performed with 6 ml of 0.5% Oil red O (Sigma) in isopropanol added to 4 ml deionized water.
Chondroblastic differentiation was tested in pellet cultures with 1 × 106 cells in 4 ml DMEM containing 1 mM sodium pyruvate (Applichem), 20 mM HEPES (Roth) pH 7.3, 0.1 µM dexamethasone, 0.1 mM 2-phospho-L-ascorbic acid, and 10 ng/ml TGF-β1 (R&D). After 4 weeks, protein extraction and western blot analysis were performed.
3
Adipogenesis Induction Protocol
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Dulbecco's modified Eagle's (DMEM) medium was from Corning Inc. (Manassas, VA, USA). Fetal bovine serum (FBS) was from GeneMate (Kaysville, UT, USA). Dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), rosiglitazone and Tam were purchased from Cayman chemical (Ann Arbor, MI, USA). Penicillin/streptomycin (P/S) was from GE Healthcare Life Sciences HyClone Laboratories (Logan, UT, USA). Insulin and NAC were from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was from Caisson Laboratories, Inc. (North Logan, UT, USA).
4
Adipocyte Differentiation Signaling Pathways
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Insulin, 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone were purchased from CAYMAN CHEMICAL COMPANY. Dimethyl sulfoxide (DMSO) and cycloheximide (CHX) were purchased from Nacalai Tesque (Tokyo, Japan). MG132 was purchased from Calbiochem (San Diego, CA, USA). An anti-phospho-MEK1/2 antibody (S217/221), anti-MEK1/2 antibody, anti-phospho-ERK1/2 antibody (T202/Y204), anti-ERK1/2 antibody, anti-phospho-Akt antibody (S473), anti-Akt antibody, anti-phospho-Insulin receptor β (IRβ) antibody (Y1146), anti-IRβ antibody, anti-phospho C/EBPβ antibody (T235), anti-Insulin receptor substrate 1 (IRS1) antibody, and anti-IRS2 antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). An anti-C/EBPδ antibody, anti-C/EBPβ antibody, anti-C/EBPα antibody, anti-PPARγ antibody, and anti-β-actin antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
5
3T3-L1 Preadipocyte Differentiation Protocol
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3T3-L1 preadipocytes were provided by the American Type Culture Collection. The cells were grown in a culture medium containing Dulbecco's modified Eagle's medium (DMEM; WELGENE, Gyeongsan-si, Korea), 10% fetal bovine serum (FBS; Hyclone Laboratories Inc., Logan, UT, USA), and 1% penicillin/streptomycin (PS; Life Technologies, Camarillo, CA, USA). The cells were cultured at 37°C in an incubator containing a humidified atmosphere of 5% CO2. 3T3-L1 cells were seeded at a density of 1.0 × 105 cells per well onto 12-well culture dishes. After 24 h, the medium was changed with an adipogenic induction medium (IM) containing minimum essential medium-alpha (α-MEM; Hyclone Laboratories Inc.), 10% FBS, 1% PS, 1 μM dexamethasone (Sigma Aldrich, St. Louis, MO, USA), 0.5 mM 3-isobutyl-1-methylxanthine (Cayman Chemical, Ann Arbor, ML, USA), 100 μM indomethacin (Sigma Aldrich), 10 mg/mL insulin (cell application, San Diego, CA, USA), and each of the three extracts at various concentrations. After four days, the medium was replaced with an adipogenic maintenance medium (MM) containing α-MEM, 10% FBS, 1% PS, 10 mg/mL insulin, and each extract. The medium was replaced every two days during the adipogenic differentiation.
6
Dexamethasone-Induced C2C12 Myotube Assay
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Water-soluble dexamethasone (Dex) from Sigma Aldrich (St. Louis, MO) was dissolved in water and added to C2C12 myotubes at a final concentration of 100 nM. Dex treatments were initiated starting on Day 5 for 24 h incubations and on Day 6 for shorter (≤12 h) incubations; all cells, control and treatment, were harvested on Day 6. All control cells were incubated with an equal volume of vehicle only. Milrinone was purchased from Selleckchem (Houston, TX, USA); Rolipram and 3-isobutyl-1-methylxanthine (IBMX) were from Cayman Chemical (Ann Arbor, MI, USA); LY294002 and H89 were from Sigma Aldrich (St. Louis, MO, USA); PKI 14–22 amide was from Tocris (Minneapolis, MN, USA). All compounds except Dex were dissolved in DMSO and added to cells at final concentrations of 10 μM for Milrinone, 25 μM for Rolipram, 250 μM for IBMX, 25 μM for LY294002, 10 μM for H89, and 10 μM for PKI 14–22 amide; the treatment times are indicated in the figure legends. When inhibitors were co-incubated with dexamethasone, they were added 15 min prior to Dex treatment.
7
Binding Assays for G-Protein Coupled Receptors
8
Adipogenesis Assay for FGF-1 and BMP-4 Bioactivity
9
Adipocyte Differentiation Reagents
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Oxysterols, oil red O stain and GW3965 were purchased from Sigma-Aldrich (St Louis, MO, USA). Dexamethasone (DEX), Insulin and 3-Isobutyl-1-methylxanthine (IBMX), cyclopamine and purmorphamine were purchased from Cayman chemical company (Ann Arbor, MI, USA). Troglitazone was purchased from Tocris Bioscience (Ellisville, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), Fetal bovine serum (FBS), Penicilin, streptomycin and L-glutamate were purchased from Mediatech, Inc (Manassas, VA, USA).
10
Vasodilator Signaling Pathway Compounds
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Ultrapure water (Milli-Q, Millipore), S-nitroso-N-acetyl-DL-penicillamine (SNAP), sodium persulfide (Na2S2, sodium disulfide; Sage Chemical Co., Ltd, Hangzhou, China), p-methoxyphenyl-morpholino-phosphinodithioic acid (GYY4137), 3-isobutyl-1-methylxanthine (IBMX) and 3'-methoxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6'-yl 2-(pyridin-2-yldisulfanyl)benzoate (Washington State Probe-1, WSP-1) from Cayman Chemicals (Biomol, Hamburg, Germany) was used. Unless otherwise specified, all other chemicals were of the highest purity available and purchased from Sigma-Aldrich (Schnelldorf, Germany or Gillingham, Dorset, UK), cell culture plastics from Greiner (Frickenhausen, Germany), and other cell culture material from PAA (Pashing, Austria). Fetal bovine serum (FBS) was from Cambrex (Lonza, Cologne, Germany).
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