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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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3 protocols using 3 isobutyl 1 methyl xanthine (ibmx)

1

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.
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2

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.
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3

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).
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