3 isobutyl 1 methyl xanthine (ibmx)

Manufactured by Topscience

Sourced in China

IBMX is a laboratory equipment product that functions as a selective phosphodiesterase (PDE) inhibitor. It is commonly used in research and scientific applications to modulate cellular signaling pathways involving cyclic nucleotides.

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Lab products found in correlation

Dexamethasone (dex), by Solarbio (2 mentions) Insulin, by Topscience (2 mentions) Lipo2000, by Thermo Fisher Scientific (1 mentions) Metformin, by Beyotime (1 mentions) Dexamethasone (dex), by Topscience (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Gentlemacs, by Miltenyi Biotec (1 mentions)

3 protocols using 3 isobutyl 1 methyl xanthine (ibmx)

Adipocyte Differentiation Protocol

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The frozen 3T3-L1 adipose precursor cells (Shanghai Bio Leaf Biotech Co., Shanghai, China) were resuscitated, passaged, and plated. Take a 6-well plate as an example, Transfection is performed when the cell density reaches about 75%. About 6 h before transfection, we changed the medium to a serum-free medium without dual antibody. We added 2 μg of recombinant plasmid and 2 μL of transfection reagent to the anti-serum-free medium, vortex to mix, and let stand for 20 min. We added the mixed solution to a petri dish, put it in the incubator for 6 h, and then replaced it with normal culture medium. After 48 h, we added induction solution I (IBMX: 0.5 mmol/L, TOPSCIENCE, Shanghai, China; Dex: 1 μmol/L, Solarbio, Beijing, China; insulin: 10 μg/mL, TOPSCIENCE, Shanghai, China) for two days, then added induction solution II (insulin: 10 μg/mL) for 8 days. Every two days, we changed the induction liquid II once. After 8 days, the cells were collected for further processing.

Liu Y., Li Y., Liang J., Sun Z., Wu Q., Liu Y, & Sun C. (2021). The Mechanism of Leptin on Inhibiting Fibrosis and Promoting Browning of White Fat by Reducing ITGA5 in Mice. International Journal of Molecular Sciences, 22(22), 12353.

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Adipocyte Differentiation and Transfection

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The cryopreserved 3T3-L1 adipocytes were recovered, passaged and planked. Taking the 6-well plate as an example, when the cell density reached about 90%, after contact inhibition for 2 days, inducible solution I (IBMX: 0.5 mmol/L, TOPSCIENCE, Shanghai, China; Dex: 1 μmol/L, Solarbio, Beijing, China; insulin: 10 μg/mL, TOPSCIENCE, Shanghai, China) was added to the culture for 2 days, then inducible solution II (insulin: 10ug/mL) was added to the culture for 8–10 days, and inducible solution II was changed every 2 days. After that, the medium was replaced with 10% serum double antibody-free medium, and the cells were transfected after 24 h of culture. We added 2 μg of recombinant plasmid and 2 μL of lipo2000 (Invitrogen, Waltham, MA, USA) to the Opti (Thermo Fisher, Shanghai, China), mixed them by vortexing, and let the solution stand for 20 min. We added the mixed solution to a Petri dish, put it in the incubator for 6 h and then replaced it with normal culture medium. After 24 or 48 h, cells were collected for further processing.

Liu Y., Li Y., Liang J., Sun Z, & Sun C. (2022). Non-Histone Lysine Crotonylation Is Involved in the Regulation of White Fat Browning. International Journal of Molecular Sciences, 23(21), 12733.

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Isolation and Cultivation of Human Adipocytes

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Human adipocytes were acquired from fresh omentum of ovarian cancer patients undergoing surgery. Fresh omentum was minced with scissors, digested by gentleMACS (Miltenyi) [45 (link)] to obtain single cells. After centrifuge with condition of 100rpm, the suspension separated into 4 layers, which are cell pallets, conditional medium, adipocytes and free fat oil. And then we carefully isolated adipocytes, counted and cultured in six-well plates with DF12 (Gibco) supplemented with 2% FBS (Gibco) for 1×10⁶ adipocytes. 48 hours later, the supernatant was harvested as conditioned medium of human primary cultured adipocytes (hCM). hCM were filtered through 200-mesh sieves to remove cell debris before use.
3T3-L1 cells were chemically induced by DMEM with 10% FBS (Gibco), 0.5mM IBMX (TOPSCIENCE, T1713), 1μM Dexamethasone (TOPSCIENCE, T1076), 5μg/ml insulin (Bovine, Sigma I-5500) for 2 days and DMEM with 10% FBS (Gibco), 10μg/ml insulin (Bovine, Sigma I-5500) for 2 days to differentiate into adipocyte-like cells. Then the supernatant of induced adipocyte-like 3T3-L1 cells was collected at 24 hours as mouse conditioned medium (mCM). After pretreatment with metformin (1 mM, Beyotime, catalog number s1741–1g) for 12 hours, the induced adipocyte-like 3T3-L1 cells were cultured with complete medium for 24 hours, and the supernatant collected as CM (MET).

Sun C., Li X., Guo E., Li N., Zhou B., Lu H., Huang J., Xia M., Shan W., Wang B., Li K., Weng D., Xu X., Gao Q., Wang S., Hu J., Lu Y., Mills G.B, & Chen G. (2019). MCP-1/CCR-2 axis in adipocytes and cancer cell respectively facilitates ovarian cancer peritoneal metastasis. Oncogene, 39(8), 1681-1695.

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