After reaching 90% cell confluence, the basic medium was removed and replaced with differentiation medium [0.25 μM dexamethasone (Takara, Dalian, China), 10 μg/ml insulin (Takara), and 0.5 mM IBMX (Takara)] for 48 h. Then, the differentiation medium was replaced with maintenance medium [10 μg/ml insulin (Takara)] and incubated for 48 h. The detailed procedure for the induction of intramuscular preadipocytes is described in Fig 1 . Cells were collected 0, 48, 96, and 144 h after induction (referred to as stages I0, I2, I4, and I6, respectively). Each stage included three biological replicates (n = 3).
Insulin
Manufactured by Takara Bio
Sourced in Japan, China
Insulin is a laboratory product used to measure insulin levels in biological samples. It provides a quantitative assessment of insulin concentrations, which is essential for research and diagnostic applications related to glucose metabolism and endocrine disorders.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Takara Bio (3 mentions)
3 isobutyl 1 methylxanthine, by Takara Bio (2 mentions)
Hydrocortisone, by Takara Bio (2 mentions)
Penicillin streptomycin, by Takara Bio (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Takara Bio (2 mentions)
Mcf 12a, by Merck Group (2 mentions)
Horse serum, by Takara Bio (2 mentions)
Human epidermal growth factor egf, by Takara Bio (2 mentions)
Human epidermal growth factor, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Insulin, by Merck Group (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Dmem ham s f 12 medium, by Corning (2 mentions)
Mcf 10a, by American Type Culture Collection (2 mentions)
Skbr3, by American Type Culture Collection (2 mentions)
Mda 361, by American Type Culture Collection (2 mentions)
Adipoinducer reagent, by Takara Bio (1 mentions)
6 well plate, by Thermo Fisher Scientific (1 mentions)
Mouse 3t3 l1 preadipocytes, by American Type Culture Collection (1 mentions)
7 protocols using insulin
1
Intramuscular Preadipocyte Differentiation
2
Preadipocyte Differentiation Protocol
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After achieving 90% cell confluence, the cells were passaged to 12-well plates and cultured until achieving 90% confluence yet again. The basic medium was then removed and replaced with differentiation medium (0.25 µM dexamethasone, 10 µg/ml insulin, and 0.5 mM 3-isobutyl-1-methylxanthine; all from Takara Bio Inc.) for 48 hr. The differentiation medium was replaced with maintenance medium (10 µg/ml insulin; Takara Bio Inc.) and incubated for 48 hr. The detailed procedure for induction of abdominal preadipocytes is outlined in Figure 1 . Cells were collected after being induced for 0, 48, 96, and 144 hr (0, 2, 4, and 6 d). Each interval included three biological replicates (n = 3).
3
Adipogenesis Induction in 3T3-L1 Cells
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Mouse 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (ATCC, Manassas, VA, U.S.A.) and cultured in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO2. To differentiate 3T3-L1 into mature adipocytes, the cells were seeded into 6-well plates (Nunc, Roskilde, Denmark) at a concentration of 8 × 104 cells per well, and the medium was replaced after 2 days. At 2 days after confluence, 3T3-L1 cells were transferred to adipogenic differentiation medium, which is DMEM containing 10% FBS and AdipoInducer Reagent (10 μg/ml insulin, 2.5 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine; Takara Bio Inc., Shiga, Japan), for 2 days. After that, the cells were cultured in adipocyte maintenance medium, which is DMEM containing 10% FBS and 10 μg/ml insulin, for 2 days. To evaluate the effect of HODE isomers in 3T3-L1 cells, the cells were cultured in the medium containing 12 μg/ml HODEs throughout the experiment.
4
Adipocyte Lipolysis and FA Uptake Assay
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Epididymal fat pads were minced and digested using type II collagenase (Sigma-Aldrich) dissolved in PBS at 37°C for 20 min on an orbital shaker. The digested tissues were centrifuged at 280 × g for 5 min. The infranatant containing the collagenase solution was removed, and the floating layer of adipocytes was washed three times with PBS. For lipolysis assay, aliquots of isolated adipocytes were incubated in the absence or presence of 100 ng/ml TNFα (R&D systems Inc., Minneapolis, MN, USA) for 3 h. Glycerol content in the infranatant medium was measured using a glycerol colorimetric assay kit (Cayman chemical). To investigate FA uptake, isolated adipocytes were incubated with 20 μM BODIPY FL C16 (Thermo Scientific) in DMEM containing 1%FA-free BSA (Sigma) in the absence or presence of 100 nM insulin (Takara Bio Inc., Shiga, Japan) for 1 h. After the transport period, cells were washed with PBS and then lysed in 0.05N NaOH (32 (link)). Fluorescence intensity was measured using FlexStation 3 multi-mode microplate reader (Molecular Devices, LLC. San Jose, CA, USA) at Ex 503 and Em 512 nm, and normalized to intra-droplet fluorescence intensity.
5
Angiogenic Factors and Signaling Pathways
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Recombinant human IL-17A homodimer, human IL-17A/F heterodimer, human basic fibroblast growth factor (bFGF), mouse antihuman IL-17RA monoclonal antibody (mAb) and goat anti-human IL-17RC polyclonal antibody (Ab) were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant human vascular endothelial growth factor (VEGF) and Suramin were purchased from KURABO (Osaka, Japan). Specific chemical inhibitors LY294002, PD98059 and SP600125 were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary HMVECs were purchased from KURABO (Osaka, Japan), and maintained in HuMedia-EB2 with 10 ng/mL epidermal growth factor (EGF), 1 μg/ mL hydrocortisone, 5 ng/mL bFGF, 10 μg/mL heparin, 39.3 μg/mL dbcAMP, 50 μg/mL gentamycin, 50 ng/mL amphotericin-B and 5% fetal calf serum (FCS) (all from KURABO, Osaka, Japan). Cells were grown at 37°C in an atmosphere of 5% CO 2 and used between passages 4 and 5. Primary human dermal fibroblasts were purchased from Takara Bio (Kusatsu, Shiga, Japan), and maintained in Fibroblast Basal Medium supplemented with 1 ng/mL bFGF, 5 μg/mL Insulin and 2% FCS (Takara Bio).
6
Culture and Maintenance of Breast Cancer Cell Lines
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Human breast cancer cell lines MDA361, T47D, SKBR3, MDA-MB-468 (MDA-468), MCF-12A, and MCF-10A were purchased from American Type Culture Collection (ATCC). MDA361, T47D, MDA-468, and SKBR3 were cultured in DMEM/F-12 (Mediatech Inc.) supplemented with 10% FBS and penicillin/streptomycin. HMLE (kindly provided by Dr. R. A. Weinberg) cell lines were cultured in 1:1 Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F-12 medium (Mediatech Inc.) supplemented with 5% FBS (Clontech), 100 units/ml penicillin-streptomycin (Invitrogen), 2 mml-glutamine (Invitrogen), 10 ng/ml human epidermal growth factor (EGF) (Invitrogen), 0.5 μg/ml hydrocortisone (Sigma), and 10 μg/ml insulin (Sigma).MCF-12A and MCF-10Awere cultured in 1:1 Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F-12 medium (Mediatech Inc.) supplemented with 5% Horse serum (Clontech), 100 units/ml penicillin-streptomycin, 10 ng/ml human epidermal growth factor (EGF), 0.5 μg/ml hydrocortisone, and 10 μg/ml insulin .
7
Culture and Maintenance of Breast Cancer Cell Lines
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