The intracellular melanin levels were determined as previously described [19 (link),67 (link)] with some modifications. B16 cells were seeded in 6-well plates at a concentration of 2.5 × 105 cells/well and allowed to attach for 24 h. This assay was divided into 7 groups as follows: control, non-treatment; IBMX: 50 µM IBMX (PanReac AppliChem, Barcelona, Spain); theophylline: IBMX + 0.01 mg/mL theophylline (Sigma Chemical, St. Louis, MO, USA); arbutin: IBMX + 0.01 mg/mL arbutin (Sigma Chemical, St. Louis, MO, USA); PES1CMU-RBO: IBMX + 0.01 mg/mL rice bran oil; PES1CMU-DFRB: IBMX + 0.01 mg/mL de-oiled rice bran extract; and PES1CMU-H: IBMX + 0.01 mg/mL husk extract. After 48 h of incubation, cell pellets were collected and lysed with 1 N NaOH containing 10% DMSO at 80 °C for 30 min. The intracellular melanin release was measured at 405 nm using a microplate reader. The results were expressed as a fold change in melanin content compared to the control.
3 isobutyl 1 methyl xanthine (ibmx)
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, France, China, Denmark, Japan, New Zealand, Macao, Sweden, Spain
IBMX is a laboratory product manufactured by Merck Group. It is a chemical compound that functions as a phosphodiesterase inhibitor. The core function of IBMX is to inhibit the activity of phosphodiesterase enzymes, which are involved in the regulation of cellular processes. This product is intended for use in research and laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (202 mentions)
Insulin, by Merck Group (149 mentions)
Indomethacin, by Merck Group (58 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (56 mentions)
Forskolin, by Merck Group (53 mentions)
Rosiglitazone, by Merck Group (37 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (30 mentions)
Oil red o, by Merck Group (29 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (24 mentions)
Ascorbic acid, by Merck Group (14 mentions)
Bovine serum albumin (bsa), by Merck Group (13 mentions)
Fetal bovine serum (fbs), by Merck Group (12 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (11 mentions)
Penicillin streptomycin, by Merck Group (11 mentions)
Penicillin, by Thermo Fisher Scientific (11 mentions)
Streptomycin, by Thermo Fisher Scientific (11 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (11 mentions)
Dmem f12, by Thermo Fisher Scientific (11 mentions)
β glycerophosphate, by Merck Group (9 mentions)
Rosiglitazone, by Cayman Chemical (9 mentions)
463 protocols using 3 isobutyl 1 methyl xanthine (ibmx)
1
Melanin Content Quantification in B16 Cells
2
Antioxidant Effects on Oocyte Maturation
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Oocytes at GV stage were divided into three groups: (1) Antioxidants group: oocytes were exposed to pre-maturation aging in M16/M2 medium (Sigma) containing 0.5 mM IBMX (Sigma, St. Louis, MO, USA) and 10 μmol sodium citrate (Sigma), 25 μmol ALA (Sigma) and 20 μmol ALCAR (Sigma); (2) Control group: oocytes were exposed to pre-maturation aging in M16/M2 medium with 0.5 mM IBMX but without any antioxidant; (3) Fresh group: oocytes were cultured in M16/M2 media for IVM without aging. For pre-maturation aging, oocytes in antioxidant group and control group were cultured in 20 μl of droplets at 37 °C in a humidified atmosphere of 5.0% CO2 for 12, 24, and 36 h, respectively. After pre-maturation aging, oocytes were cultured without IBMX for 14 h in M2 medium supplemented with 10% FBS, recombinant 75 mIU/mL follicle-stimulating hormone (Sigma), 0.5 IU/mL human chorionic gonadotropin (Sigma), and 5 ng/mL recombinant epidermal growth factor (Sigma). Oocytes in fresh group were directly cultured for 14 h in the IVM medium without pre-maturation aging. After IVM, oocytes with extrusion of the first polar body were considered as MII stage. Oocytes at MII stage were counted and then used for assessment of mitochondrial distribution and spindle morphology. The oocytes arrested at GV stage after IVM were used for examination of DNA DSBs.
3
cAMP Accumulation Assay in 3T3-L1 Cells
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Cyclic AMP accumulation assay was performed in 96-well plates 2 days post confluence of 3T3-L1 cells. In brief, cells were washed in serum-free DMEM containing 1 mM IBMX (Sigma-Aldrich) and further incubated for 15 min in 100 µl serum-free DMEM containing 1 mM IBMX and the respective compounds. Cells were lysed using LI buffer (5 mM HEPES, 0.3% Tween-20, 0.1% BSA, and 0.5 mM IBMX). The amount of cAMP was determined using the AlphaScreenTM cAMP Functional Assay (PerkinElmer, Rodgau, Germany) according to the manufacturer’s protocol using the EnVision 2105 Multimode Plate Reader (PerkinElmer, Rodgau, Germany).
4
Isolation and Manipulation of Mouse Oocytes
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Fully grown GV-stage oocytes were isolated from the ovaries of C57BL/6 female mice following hormonal priming with 7.5 IU of pregnant mare serum gonadotrophin (Intervet). Oocytes were handled in a Petri dish containing prewarmed M2 medium (Sigma-Aldrich) at 37°C supplemented with 100 µM of the phosphodiesterase inhibitor, IBMX (Sigma-Aldrich), which prevents oocytes from undergoing GVBD. Only fully grown cumulus-covered oocytes with clearly identifiable GVs were used in experiments. Surrounding cumulus cells were mechanically denuded using mouth pipetting. To enable maturation, denuded GV-stage oocytes were washed through multiple droplets of M16 medium (Sigma-Aldrich) to remove all traces of IBMX. For longer-term culture, groups of oocytes were placed in dishes containing M16 microdroplets covered with mineral oil (Sigma-Aldrich) and incubated at 37°C in an atmosphere of 5% CO2 in air.
DNA DSBs were induced by incubating oocytes in M16 medium containing either Eto (Sigma-Aldrich; 10 µg/ml) or doxorubicin (Hospira; 10 µg/ml) for 3 h or by exposure to UV-B radiation via a UV transilluminator (Vilber Lourmat; 312 nm) for 5 s. ROS production was attenuated by incubating oocytes overnight in M16 medium supplemented with NAC (5 mM; Abcam 139476). The 26S proteasome inhibitor, MG132 (SelleckChem; 5 µM), was used to inhibit proteolysis as described previously (Wei et al., 2018 (link)).
DNA DSBs were induced by incubating oocytes in M16 medium containing either Eto (Sigma-Aldrich; 10 µg/ml) or doxorubicin (Hospira; 10 µg/ml) for 3 h or by exposure to UV-B radiation via a UV transilluminator (Vilber Lourmat; 312 nm) for 5 s. ROS production was attenuated by incubating oocytes overnight in M16 medium supplemented with NAC (5 mM; Abcam 139476). The 26S proteasome inhibitor, MG132 (SelleckChem; 5 µM), was used to inhibit proteolysis as described previously (Wei et al., 2018 (link)).
5
Adipocyte Differentiation Protocol
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For collected iWAT samples, the connective fiber and blood vessels were removed and washed three times with PBS buffer containing 200 U/mL penicillin (Sigma, St. Louis, USA) and 200 U/mL streptomycin (Sigma, St. Louis, USA). The adipocyte culture was carried out according to our previous publication. Briefly, pre-adipocytes were seeded onto 35-mm culture dishes at a density of 8 × 104 cells/dish, and incubated at 37 °C under a humidified atmosphere of 5% CO2 and 95% air until confluence. Differentiation of white pre-adipocytes was performed as following. Cells grown to 100% confluence (Day 0) were exposed to the induction DMEM/F12 containing dexamethasone (1 μM; Sigma), insulin (10 μg/mL; Sigma), IBMX (0.5 mM, Sigma) and 10% FBS. Four days after the induction (from Day 2), cells were maintained in the induction medium containing insulin (10 μg/mL, Sigma) and 10% FBS until the day for cell harvest. And brown adipocytes were induced to differentiate using DEME/F12 with 15% FBS, 0.5 mM IBMX; 0.25 mM indomethacin; 2 μg/mL dexamethasone; 1 nM T3, 20 nM insulin for 2 days and subsequently maintained on differentiation media (1 nM T3, 20 nM insulin).
6
Adipocyte Differentiation Protocol
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Cell lines, including 3T3-L1 preadipocytes and 293T, were cultured in normal growth media consisting of Dulbecco’s modified Eagle’s medium (Hyclone, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Hyclone, Thermo Fisher Scientific). All cells were grown at 37°C in a humidified atmosphere of 5% CO2. To induce white adipocytes, 3T3-L1 preadipocytes were treated with differentiation medium I (DMI) from days 2 to 5 (postconfluence designated as day 0). DMI is composed of normal growth media, insulin (1 μg/ml; Sigma), dexamethasone (1 μM; Sigma), and 3-isobutyl-1-methyl-xanthine (0.5 mM; IBMX, Sigma). Cells were then switched to DMII, which includes normal growth medium and insulin (1 μg/ml), from days 5 to 9, after which they were placed back into normal growth medium and refed every 2 days (32 (link)). To induce beige adipocytes, 0.5 μM rosiglitazone (Rosi, Sigma) and 0.5 mM IBMX were added into DMII, and then, the cells were exposed to 10 μM isoproterenol (Iso, Sigma) for 4 hours before harvesting (33 (link), 34 (link)).
7
Isolation and Maturation of Mouse Oocytes
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Four- to six-week-old female Kunming mice were sacrificed by cervical dislocation 48 h after intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG; Solarbio, P9970). Fully grown GV stage oocytes were collected by ovarian puncture in a prewarmed F12 medium (Ham, YC-3034) supplemented with 50 μM IBMX (Sigma, 15879, St Louis, MO, USA), and were then used for culture or microinjection. For meiotic maturation, oocytes were washed three times to remove IBMX and then cultured in a prewarmed MII (Sigma, M7167) medium covered with paraffin oil (Sigma, M8410) at 37 °C in a 5% CO2 atmosphere. Different stages of oocytes were collected for the following experiments.
8
Harvesting Mouse Oocytes for IVM
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Fully-grown GV oocytes were obtained from the ovaries of superovulated female mice 48 h after intraperitoneal injection of 7.5 IU of pregnant mare serum gonadotropin (PMSG) as described previously37 (link). Ovaries were placed in a Petri dish with M2 medium (Sigma-Aldrich) supplemented with IBMX (Sigma-Aldrich) to prevent oocytes from undergoing GVBD. GV oocytes were released by puncturing antral follicles with a fine needle under a dissecting microscope. To collect MII-stage oocytes, 7.5 IU of human chorionic gonadotropin (hCG) was administered 48 h after PMSG injection to induce ovulation. Mice were sacrificed 15– 16 h post hCG by cervical dislocation and cumulus-oocyte complexes (COCs) were released from the oviducts. COCs were briefly incubated in M2 medium containing 0.3 mg/mL hyaluronidase (Merck Millipore) to remove cumulus cells. For in vitro maturation, oocytes were washed and cultured in IBMX-free M16 medium (Sigma-Aldrich) for various periods of time at 37 °C in 5% CO2 atmosphere.
9
Assaying cAMP Accumulation
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A biochemical assay of cAMP accumulation was used to determine the effects of AC mutation on catalytic activity, with high sensitivity and without dependence on subcellular location due to inhibition of cellular phosphodiesterases. Briefly, cells were pre-incubated in the presence of 1 mM IBMX (Sigma) for 30 min at 37°C in Dulbecco's modified Eagle's medium followed, and then incubated for an additional 10 min in absence or presence of isoproterenol (in the continued presence of IBMX), as indicated. Cells were quickly washed with ice-cold PBS and lysed by exposure to 0.1 M HCl for 10 min at room temperature. The cAMP concentration in lysates was determined using a commercial immunoassay (Direct cAMP ELISA kit, Enzo Life Sciences, Farmingdale, NY) according to the manufacturer’s instructions.
10
Preadipocyte Isolation and Adipogenic Induction
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At 3 days old piglets were sacrificed, the intramuscular preadipocytes in LD were extracted as previously described [32 (link)]. Cells were re-suspended in DMEM/F12 and plated at a density 6 × 105 per 60-mm culture dish (Fig. S1 A), and cultured in a 5% CO2 incubator at 37 °C. When the cells grow to confluence (Fig. S1 C), the medium was changed with adipogenic induction medium, which is the DMEM/F12 supplement with 10% FBS, 100 U/mL penicillin-streptomycin, 0.5 mmol/L IBMX, 1 nmol/L DEX, and 5 ng/mL insulin (IBMX, DEX and insulin were purchased from Sigma).
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