Methylisobutylxanthine ibmx

Manufactured by Merck Group

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Methylisobutylxanthine (IBMX) is a laboratory reagent used in cell biology and biochemical research. It is a phosphodiesterase inhibitor, which means it blocks the breakdown of cyclic nucleotides, such as cAMP and cGMP, in cells. IBMX can be used to study the effects of increased intracellular levels of these signaling molecules.

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Methylisobutylxanthine (33 citations)

8 protocols using methylisobutylxanthine ibmx

Insulin-stimulated Glucose Uptake in Differentiated 3T3-L1 Adipocytes

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We differentiated 3T3-L1 cells into adipocytes, by stimulating 1 × 10⁴ cells in a black 96-well plate with DMEM (Life Technologies) media containing 25 mM glucose (3.4 g L⁻¹ sodium bicarbonate, 50 U mL⁻¹ penicillin and streptomycin and 10% heat-inactivated FBS), 0.5 mM methylisobutylxanthine (IBMX; Sigma), 1 μM dexamethasone (Sigma) and 10 μg mL⁻¹ insulin (Sigma) for 2 days. This media was then replaced with fresh media containing 10 μg mL⁻¹ insulin for a further 2 days. Following this, media was replaced with fresh media every 48 h, until adipocyte differentiation.
All experiments were carried out on day 8 post-differentiation. Cells were cultured in serum-free 2.8 mM glucose media for 2 h before the addition of insulin. We treated cells for 1 h with 2 ng ml⁻¹ recombinant insulin, proinsulin, or total insulin from islet secretions at 37 °C as described in figure legends. Subsequently, cells were incubated with 10 μM 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-6-deoxyglucose (2-NBDG; Molecular Probes) for 20 min before washing extensively with PBS and determining glucose uptake using a POLARstar Omega plate reader.

Sajiir H., Wong K.Y., Müller A., Keshvari S., Burr L., Aiello E., Mezza T., Giaccari A., Sebastiani G., Dotta F., Ramm G.A., Macdonald G.A., McGuckin M.A., Prins J.B, & Hasnain S.Z. (2024). Pancreatic beta-cell IL-22 receptor deficiency induces age-dependent dysregulation of insulin biosynthesis and systemic glucose homeostasis. Nature Communications, 15, 4527.

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Adipocyte Differentiation of 3T3-L1 Preadipocytes

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The 3T3-L1 preadipocytes were cultured as previously described^{12 (link),13 (link)}. The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% (v/v) calf serum (BCS; Gibco), 100 µg/mL penicillin, and 100 µg/mL streptomycin in a 5% CO₂ incubator at 37 °C. The cells were infected with lentivirus and adipocyte differentiation was induced after 48 h. For adipocyte differentiation, confluent cells were induced to differentiate in DMEM containing 10% fetal bovine serum (FBS; Gibco) and MDI (520 µM methylisobutylxanthine (IBMX; Sigma-Aldrich), 1 µM dexamethasone (Sigma-Aldrich) and 167 nM insulin(Gibco)). After 2 days, the medium was changed with DMEM containing 10% FBS and insulin. On day 4, the medium was replaced with DMEM containing 10% FBS and changed with fresh identical medium every 2 days for up to day 8. On day 8, cells were harvested for further analyses.

Park J.C., Jeong W.J., Seo S.H, & Choi K.Y. (2019). WDR76 mediates obesity and hepatic steatosis via HRas destabilization. Scientific Reports, 9, 19676.

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Adipogenesis Induction and Cell Treatment

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3T3-L1 cells were derived from the American Type Culture Collection (CL-173, Manassas, VA, USA). Culture of 3T3-L1 cells and adipogenesis induction were performed as previously described (Lee et al., 2015 (link)) with modifications. Cells were cultured in pre-adipocyte expansion medium based on Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). Cells were seeded at a density of 8×10⁴ cells/well in 24-well culture plates in triplicate (n=3). Confluent cells were cultured in adipocyte differentiation cocktail medium [0.5 mM methylisobutylxanthine (IBMX, Sigma), 1 μM dexamethasone (Sigma), and 1 μg/mL insulin (Sigma) in DMEM supplemented with 10% fetal bovine serum (FBS)] for 4 days, then in adipocyte maintenance medium (DMEM supplemented with 10% FBS and 1 μg/mL insulin) for 2 days, followed by DMEM supplemented with 10% FBS for a further 8 days. Cells were then cultured in a CO₂ incubator at 37°C for adipocyte differentiation to complete. Differentiated adipocytes were treated with 1% MRS as a blank, or 1% culture supernatant of TCI378 without cells, and cultured in a CO₂ incubator at 37°C for 2 days.

Huang C.H., Ho C.Y., Chen C.T., Hsu H.F, & Lin Y.H. (2019). Probiotic BSH Activity and Anti-Obesity Potential of Lactobacillus plantarum Strain TCI378 Isolated from Korean Kimchi. Preventive Nutrition and Food Science, 24(4), 434-441.

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PLGA Microparticles for Adipogenic Differentiation

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75:25 poly (D,L-lactide-co-glycolide) (PLG) with a lauryl ester end group and an inherent viscosity of 0.79 dL/g was purchased from Evonik (Birmingham, AL). Rosiglitazone was purchased from Cayman Chemical (Ann Arbor, MI). Dichloromethane (DCM) and polyvinyl alcohol (PVA, Mw 13,000–23,000, 87–89% hydrolyzed) were purchased from Sigma (St. Louis, MO). Ethyl acetate was purchased from Macron Fine Chemicals (Center Valley, PA). 200 proof ethanol was purchased from Decon Laboratories (King of Prussia, PA). Dimethyl sulfoxide (DMSO) was purchased from Fisher (Hampton, NH). Ultrapure water was obtained from a Thermo Scientific Barnstead Nanopure system. Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose and L-glutamine was purchased from Corning Cellgro (Corning, NY). Trypsin with 0.25% EDTA, Fetal Bovine Serum (FBS) and Pen/Strep were purchased from Fisher (Hampton, NH). Super Calf Serum was purchased from GemCell (Sacramento, CA). Insulin (bovine), dexamethasone, and methylisobutylxanthine (IBMX) were purchased from Sigma (St. Louis, MO). 10% Neutral Buffered Formalin was purchased from Sigma (St. Louis, MO). Nile Red was purchased from Molecular Probes (Eugene, OR). Adiponectin ELISA kit was purchased from R&D systems (Minneapolis, MN).

Spetz M.R., Isely C, & Gower R.M. (2021). Effect of fabrication parameters on morphology and drug loading of polymer particles for rosiglitazone delivery. Journal of drug delivery science and technology, 65(1-2), 10.1016/j.jddst.2021.102672.

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Adipogenic Differentiation of 3T3-L1 Cells

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The 3T3L1 preadipocytes were initially maintained in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µg/mL penicillin and 100 µg/mL streptomycin in a humidified atmosphere of 95% air/ 5% CO₂ at 37 °C. The respective nanofiber samples were ultraviolet (UV) sterilized, rinsed in phosphate buffer saline (PBS) and soaked in cell culture media overnight prior to cell seeding. The cells were seeded to nanofiber samples with a cell density of 3 × 10⁴ cells/well and incubated at conditions suitable for cell growth. After 48 hours the medium was changed to differentiation medium supplemented with 10% FBS, 100 µg/mL penicillin, 100 µg/mL streptomycin, 1 µM dexathamethasone, 0.5 mM Methylisobutylxanthine (IBMX) and 10 µg/ml insulin (Sigma Aldrich, USA). After 48 hours the differentiation medium was replaced with Adipocyte maintenance medium supplemented with 10% FBS, 100 µg/mL penicillin, 100 µg/mL streptomycin and 1 µg/ml insulin and cultured until day 14.

Unnithan A.R., Sasikala A.R., Thomas S.S., Nejad A.G., Cha Y.S., Park C.H, & Kim C.S. (2018). Strategic Design and Fabrication of Biomimetic 3D Scaffolds: Unique Architectures of Extracellular Matrices for Enhanced Adipogenesis and Soft Tissue Reconstruction. Scientific Reports, 8, 5696.

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Chemically-Induced Adipocyte Differentiation

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Cells were differentiated according to ATCC’s chemically-induced differentiation protocol. Cells were plated in pre-adipocyte expansion media. Two days post-100% confluency is designated as Differentiation Day 0 (DD0). From DD0 until DD2, cells continued to be cultured in pre-adipocyte expansion media. On DD2, media was replaced with adipocyte expansion media, containing DMEM (ATCC), 10% Fetal Bovine Serum (Sigma), 1% penicillin/streptomycin, 1.0 μM dexamethasone (Sigma), 0.5 mM methylisobutylxanthine (IBMX; Sigma) and 1.0 μg insulin (Sigma). On DD5, adipocyte expansion media was replaced with adipocyte maintenance media, containing DMEM (ATCC), 10% FBS, 1% penicillin/streptomycin and 1.0μg insulin (Sigma) until DD8.

Jacobs D.T., Allard B.A., Pottorf T.S., Silva L.M., Wang W., Al-Naamani A., Agborbesong E., Wang T., Carr D.A, & Tran P.V. (2019). Intraflagellar-transport A dysfunction causes hyperphagia-induced systemic insulin resistance in a pre-obese state. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(1), 148-160.

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Nanoparticle-Mediated Resveratrol Delivery

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75:25 poly (D,L-lactide-co-glycolide) with a lauryl ester end group and an inherent viscosity of 0.79 dL/g was purchased
from Evonik. Dichloromethane, resveratrol, poly (vinyl alcohol) (Mw 13,000-23,000, 87-89% hydrolyzed), and fluorescein
isothiocyanate were purchased from Sigma (St. Louis, MO). 200 proof ethanol was purchased from Decon Laboratories (King of
Prussia, PA). Dimethyl sulfoxide (DMSO) was purchased from Fisher (Hampton, NH). Ultrapure water was obtained from a Thermo
Scientific Barnstead Nanopure system. 3T3-L1 mouse embryonic fibroblasts (CL-173) were purchased from ATCC (Manassas, VA).
Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose and L-glutamine was purchased from Corning Cellgro
(Corning, NY). Trypsin with 0.25% EDTA, Fetal Bovine Serum (FBS) and Pen/Strep were purchased from Fisher (Hampton, NH). Super
Calf Serum was purchased from GemCell (Sacramento, CA). Insulin (bovine), dexamethasone, and methylisobutylxanthine (IBMX) were
purchased from Sigma (St. Louis, MO).

Isely C., Hendley M.A., Murphy K.P., Kader S., Annamalai P., Esmaiel J, & Gower R.M. (2019). Development of microparticles for controlled release of resveratrol to adipose tissue and the impact of drug loading on particle morphology and drug release. International journal of pharmaceutics, 568, 118469.

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Adipocyte Differentiation Protocol

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Insulin, methylisobutylxanthine (IBMX) and dexamethasone were purchased from Sigma-Aldrich (St. Louis, MO, USA). To induce adipocyte differentiation, (1) cells were cultured in DMEM medium for 2 days, (2) treated with DMEM medium containing 0.5 mM IBMX, 1 μM dexamethasone and 10 μg/mL Insulin for 3 days, and finally (3) cultured with DMEM medium containing 10 µg/mL Insulin for 3 days, as described [65 (link)]. The cells were then incubated in fresh DMEM medium for an additional 2 days and fully differentiated adipocyte-like cells were obtained.

Uchiyama T., Itaya-Hironaka A., Yamauchi A., Makino M., Sakuramoto-Tsuchida S., Shobatake R., Ota H., Takeda M., Ohbayashi C, & Takasawa S. (2019). Intermittent Hypoxia Up-Regulates CCL2, RETN, and TNFα mRNAs in Adipocytes via Down-regulation of miR-452. International Journal of Molecular Sciences, 20(8), 1960.

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