Immortalized brown preadipocytes from interscapular BAT of UCP1-mRFP1 transgenic mice were cultured as described previously.83 (link) Briefly, post-confluent immortalized primary cells were incubated in a differentiation medium containing 0.25 μM DEX (Nacalai Tesque), 0.5 mM IBMX (Nacalai Tesque), 1 nM T3 (Sigma-Aldrich), 10 μg/mL insulin (Wako Pure Chemical), 125 μM indomethacin (Wako Pure Chemical), and 0.5 μM rosiglitazone (LKT Laboratories) in the growth medium. After 48 h, the cell culture medium was replaced with a post-differentiation medium containing 5 μg/mL insulin (Wako Pure Chemical), 1 nM T3 (Sigma-Aldrich), and 0.5 μM rosiglitazone (LKT Laboratories) in the growth medium, and the medium was changed every two days. For the ISO treatment experiments, differentiation-induced brown adipocytes were stimulated with 1 μM ISO (Sigma-Aldrich) for the indicated time durations, as described in Figures S1 A and S1C and harvested for further analysis.
3 isobutyl 1 methyl xanthine (ibmx)
Manufactured by Nacalai Tesque
IBMX is a chemical compound used as a reagent in laboratory settings. Its core function is to inhibit the enzyme phosphodiesterase, which plays a role in the breakdown of the second messenger molecule cyclic AMP (cAMP) within cells. By inhibiting this enzyme, IBMX can lead to an increase in intracellular cAMP levels, which can have various effects depending on the specific experimental context.
Automatically generated - may contain errors
Lab products found in correlation
Indomethacin, by Fujifilm (1 mentions)
Insulin, by Fujifilm (1 mentions)
Dexamethasone (dex), by Nacalai Tesque (1 mentions)
Rosiglitazone, by LKT Laboratories (1 mentions)
Cyclic amp select elisa kit, by Cayman Chemical (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
2 protocols using 3 isobutyl 1 methyl xanthine (ibmx)
1
Differentiation and Thermogenic Activation of Immortalized Brown Preadipocytes
2
Intracellular cAMP Quantification Assay
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A cAMP assay was conducted by using the
cyclic AMP select ELISA kit (Cayman Chemical). The transfected HEK293
cells were seeded on cultured dishes and incubated for 2 days in growth
medium at 37 °C in a humidified atmosphere of 95% air and 5%
CO2. The cultured medium was replaced into serum-free DMEM
supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 unit/mL
penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin
B, and the cells were incubated at 37 °C for 60 min (serum-starvation).
After removal of DMEM, HBS solutions containing agonists and a phosphodiesterase
inhibitor (200 μM IBMX (Nacalai tesque)) were added to the cells
followed by incubation at 37 °C for 15 min. After HBS solutions
were removed, 0.1 M HCl solution was added to the cells, and incubated
at room temperature for 20 min. The resultant cells were collected
by cell scrapers and homogenized by pipetting. The homogenized solutions
were analyzed by the cyclic AMP select ELISA kit (Cayman Chemical)
according to the manufacture’s instruction.
cyclic AMP select ELISA kit (Cayman Chemical). The transfected HEK293
cells were seeded on cultured dishes and incubated for 2 days in growth
medium at 37 °C in a humidified atmosphere of 95% air and 5%
CO2. The cultured medium was replaced into serum-free DMEM
supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 unit/mL
penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin
B, and the cells were incubated at 37 °C for 60 min (serum-starvation).
After removal of DMEM, HBS solutions containing agonists and a phosphodiesterase
inhibitor (200 μM IBMX (Nacalai tesque)) were added to the cells
followed by incubation at 37 °C for 15 min. After HBS solutions
were removed, 0.1 M HCl solution was added to the cells, and incubated
at room temperature for 20 min. The resultant cells were collected
by cell scrapers and homogenized by pipetting. The homogenized solutions
were analyzed by the cyclic AMP select ELISA kit (Cayman Chemical)
according to the manufacture’s instruction.
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