Cells were kept in a growth medium (GM) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), which was stored in an incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO2 environment. We used a differentiation medium (DM) containing Dulbecco’s modified Eagle’s medium (DMEM; VivaCell, Shanghai, China) supplemented with 10% FBS, 1 μmol/L dexamethasone (DEX; Solarbio, Beijing, China), 0.5 mmol/L 3-isobutyl-1-methylxanthine (IBMX; Solarbio, Beijing, China), and 10 μg/mL insulin (Solarbio, Beijing, China). The maintenance medium (MM) consisted of DMEM with 10% FBS and 10 μg/mL insulin. According to the manufacturer’s instructions, transfection was carried out using the lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). The transfection reagent was mixed with RNA oligo (let-7a-5p mimic, inhibitor, negative control (NC), inhibitor negative control (INC)) and siRNA (si-Srebf2, si-Thbs1, and si-NC), and then the mixture was transfected into the cells. The concentration of transfection was 50 uM for the mimic and NC, and 100 uM for the inhibitor, INC, siRNA, and si-NC. The above RNA oligo and siRNA were purchased from Sangon Biotech Co., Ltd. (Shanghai, China) and Tsingke Biotechnology Co., Ltd. (Beijing, China), respectively, and Table S5 displays the sequence information.
3 isobutyl 1 methylxanthine ibmx
Manufactured by Solarbio
Sourced in China
3-isobutyl-1-methylxanthine (IBMX) is a xanthine derivative that functions as a nonselective phosphodiesterase inhibitor. It inhibits the enzyme activity of phosphodiesterases, which are responsible for the breakdown of cyclic nucleotides such as cAMP and cGMP within cells.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Solarbio (3 mentions)
Insulin, by Solarbio (3 mentions)
Insulin, by Merck Group (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Cck 8 kit, by Vazyme (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
4 protocols using 3 isobutyl 1 methylxanthine ibmx
1
Adipocyte Differentiation Protocol
2
Isolation and Differentiation of Duck Preadipocytes
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In a sterile environment, 42-day-old male Cherry Valley ducks were selected and killed by venous bleeding. Duck preadipocytes were isolated according to the duck cell separation and culture method presented by Ding et al. [19 (link)]. During the cell culturing, the medium was replaced every two days.
Cells were reseeded into 96-well and 6-well plates for the experiment after 80% cell confluence. According to the instructions of the CCK-8 kit (Vazyme, Nanjing, China), the cell inoculation density was 2000 cells/well (n = 3), and the growth curve of cells was detected. During the 6-day process of cell differentiation, the differentiation medium was used for the culture, and the medium was changed and detected every two days. The differentiation medium consisted of a complete medium, 0.5 mmol/L of 3-isobutyl-1-methylxanthine (IBMX, Solarbio, Beijing, China), 1 μmol/L of dexamethasone (DEX, Solarbio, Beijing, China), 10 µg/µL of insulin (Sigma, Shanghai, China), and 300 µM of oleic acid (Sigma, Shanghai, China). The optimal induction time of duck preadipocytes was on the fourth day.
Cells were reseeded into 96-well and 6-well plates for the experiment after 80% cell confluence. According to the instructions of the CCK-8 kit (Vazyme, Nanjing, China), the cell inoculation density was 2000 cells/well (n = 3), and the growth curve of cells was detected. During the 6-day process of cell differentiation, the differentiation medium was used for the culture, and the medium was changed and detected every two days. The differentiation medium consisted of a complete medium, 0.5 mmol/L of 3-isobutyl-1-methylxanthine (IBMX, Solarbio, Beijing, China), 1 μmol/L of dexamethasone (DEX, Solarbio, Beijing, China), 10 µg/µL of insulin (Sigma, Shanghai, China), and 300 µM of oleic acid (Sigma, Shanghai, China). The optimal induction time of duck preadipocytes was on the fourth day.
3
Adipocyte Differentiation Protocol
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Preadipocytes were maintained in a growth medium (GM) containing 10% fetal bovine serum (FBS, Gibco, CA, USA) in an incubator (Thermo Fisher Scientific, San Jose, CA, USA) at 37 °C and 5% CO2 environment, and the medium was changed every 24 h. When the cell density reached 70–80%, the induced differentiation medium (5% FBS, 1 μmol/L dexamethasone (DEX; Solarbio, Beijing, China), 0.5 mmol/L 3-isobutyl-1-methylxanthine (IBMX; Solarbio, Beijing, China) and 10 ug/mL insulin (Solarbio, Beijing, China)) was used to induce differentiation for 3 d to observe the lipid droplet formation of preadipocytes. Then, the medium was replaced with a maintenance medium containing 5% FBS and 10 ug/mL insulin for 3 d. Subsequently, the medium was replaced with GM to obtain mature adipocytes.
4
3T3-L1 Adipocyte Differentiation Assay
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3T3-L1 preadipocyte cells were cultured in DMEM complete medium and grown to confluence. The medium was replaced with complete medium containing 1.7 μM insulin, 1.0 μM dexamethasone and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (Solarbio, Beijing, China), and the cells were cultured for 48 h. Then, the medium was replaced with complete medium containing 1.7 μM insulin (Sigma, St. Louis, MO, USA) and cultured for an additional 48 h. Finally, the cells were maintained in complete medium containing the indicated concentrations of KLTI as Coix seed oil, and the medium was replaced with fresh medium containing the indicated concentrations of KLTI every 48 h. On day 8, the supernatant was collected and filtered to remove debris to obtain adipocyte-conditioned medium. The cells were washed twice with cold PBS (136.9 mM NaCl, 2.68 mM KCl,4 mM Na2HPO4, and 1.76 mM KH2PO4; pH 7.4), fixed in 3.7% paraformaldehyde (Solarbio, Beijing, China) for 1 h and then stained with 0.2% (w/v) Oil red O(Rowley Biochemical, USA) in a 60% isopropanol (IT7048, Shanghai Sangon Biological Engineering Co. Ltd, China) solution for 1 h. Oil red staining was observed and analyzed using an inverted microscope (Olympus, Tokyo, Japan). Stained cells were treated with 100% isopropyl alcohol to extract the Oil red O, and the colorimetric intensity of the extract was measured at 595 nm to determine the lipid content.
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