Adipocytes were incubated in Krebs Ringer Bicarbonate (KRB) buffer supplemented or not with basolateral medium from Caco-2 cells treated with either predetermined concentrations of the extracts, at a dilution of 1/8, or the pro-lipolytic reference products (Forskolin and Isoproterenol), at 37 °C for 2 h. Forskolin (1 µm, F6886-Sigma) and isoproterenol (0.1 µm, I6504-Sigma) were used as activators of lipolysis. Cell culture media were then collected and frozen at − 20 °C. The lipolytic activity of human adipocytes was assessed by the measurement of glycerol (Glycerol assay, GY105, Randox), and non-esterified fatty acid release (NEFA-HR2 R1 Set, 434-91795 et NEFA-HR2 R2 Set, 436-91995, Wako) in the adipocyte culture medium. Adipocytes were collected for DNA quantification (Quant-iT™ PicoGreen™ dsDNA Assay Kit, P7589, Invitrogen) to normalize glycerol and NEFA concentrations.
I6504
Manufactured by Merck Group
Sourced in United States
The I6504 is a laboratory equipment product from Merck Group. It is designed to perform a core function for laboratory operations. However, a detailed and unbiased description is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
F6886, by Merck Group (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
P9541, by Merck Group (1 mentions)
S7653, by Merck Group (1 mentions)
A2661, by Merck Group (1 mentions)
Alzet osmotic minipump, by Durect (1 mentions)
Atglistatin, by Cayman Chemical (1 mentions)
Etomoxir, by Bio-Techne (1 mentions)
Etomoxir, by Cayman Chemical (1 mentions)
Mp150, by Biopac (1 mentions)
D4540, by Merck Group (1 mentions)
C57bl 6j, by Jackson ImmunoResearch (1 mentions)
G 4408, by LC Laboratories (1 mentions)
P0884, by Merck Group (1 mentions)
F3917, by Merck Group (1 mentions)
F6428, by Merck Group (1 mentions)
S2929, by Selleck Chemicals (1 mentions)
Norepinephrine, by Merck Group (1 mentions)
Epinephrine, by Merck Group (1 mentions)
16 protocols using i6504
1
Adipocyte Lipolysis Measurement
2
Monitoring cAMP Dynamics in Cardiomyocytes
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Cardiomyocytes were transfected with adenoviral vectors encoding the Förster resonance energy transfer (FRET) cAMP sensor Epac-SH187 by incubation overnight at 37°C, 5% CO2 prior to the FRET measurements. Cytosolic cAMP response to different stimuli in the transfected cells was recorded as cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) emission intensity change. Cells were bathed in Tyrode solution (135 mM NaCl (Sigma, S7653), 4.5 mM KCl (Sigma, P9541), 20 mM HEPES (Sigma, H3375), 11 mM glucose (Sigma, G7021), 1 mM MgCl2 (SERVA, 39772.02), 2 mM CaCl2 (BioVision, B1010), titrated with NaOH to pH 7.4) to reach a stable baseline before isoprenaline (Sigma, I6504), IBMX (non-selective phosphodiesterase inhibitor; 3-isobutyl-1-methylxanthine, Sigma, I5879) and forskolin (FSK, adenylyl cyclase agonist; Cayman Chemical, 11018) were applied. Images were taken using a Nikon Eclipse Ti2 inverted microscope with a 40× oil immersion objective, connected to a Cairn Research dual OptoLED light source, and a Prime BSI photometric camera. Excitation wavelength was set at 430 nm, and emission was measured at 480 and 535 nm for CFP and YFP, respectively. Background fluorescence was subtracted from the recorded emission intensities, and FRET ratio was calculated as CFP over YFP and normalized to the average FRET ratio at baseline for comparison.
3
Sympathetic and Parasympathetic Responses
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Acute effects of the sympathetic agonist, isoproterenol (ISO, I6504, Sigma-Aldrich) and the parasympathetic agonist, acetylcholine (ACh, A2661, Sigma-Aldrich) were studied by videomicroscopy and calcium imaging at 48 hours post-culture. Final concentrations of 100 nM ISO and 1 μM ACh were added to the culture media to stimulate sympathetic and parasympathetic pathways, respectively. Drug effects were evaluated by videomicroscopy. In a second set of experiments, changes in spatiotemporal activity by ISO and ACh were studied using calcium imaging with fluo-4.
4
Isoproterenol-Induced Cardiac Hypertrophy
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Following initial phenotyping, adult (10-week-old) PLN-R14Δ/+ mice and their WT littermates were randomly subjected to infusion of isoproterenol (30 mg/kg/day for 4 weeks; I6504 Sigma-Aldrich) using subcutaneously implanted ALZET osmotic mini pumps (model 2004; DURECT Corporation, CA, USA) or sham surgery as previously described44 (link).
5
Adipocyte Lipolysis Regulation Protocols
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Lipase inhibitor, Triascin C, Etomoxir treatment: On day 7 of differentiation, media was aspirated and replaced with maintenance media supplemented with lipase inhibitors (10 μM Atglistatin (Cayman Chemicals, 15284) and 20 μM CAY10499 compound (Cayman Chemicals, 10007875) or 5 μM Triascin C (Sigma-Aldrich, T4540) or 50 μM Etomoxir (Tocris, 4539). After 1 h preincubation, cells were stimulated with 100 nM isoproterenol (ISO) (Sigma-Aldrich, I6504) or 20 μM SR-3420 (Rondini et al., 2017 (link)). Cells were harvested for RNA extraction after 3 h stimulation.
NE stimulation: On day 7 of differentiation, cells were stimulated with 1 μM NE (Sigma Aldrich, A9512) or sterile H2O. Cells were harvested for RNA extraction after 1 h stimulation.
NE stimulation: On day 7 of differentiation, cells were stimulated with 1 μM NE (Sigma Aldrich, A9512) or sterile H2O. Cells were harvested for RNA extraction after 1 h stimulation.
6
Isoproterenol-Induced Cardiac Fibrosis Model
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Male Sprague-Dawley rats (6-week-old) were obtained from the Shanghai Experimental Animal Center. The rats were randomly assigned to three groups (n = 10 per group): (1) control, (2) isoproterenol (ISO, 5 mg/kg/day), and (3) ISO + TMZ (15 mg/kg/day). The rats were subcutaneously injected with ISO (I6504, Sigma, St. Louis, MO, USA) for 7 days and then fed for another 14 days to establish experimental cardiac fibrosis. The control rats were similarly administered with an equal volume of saline. From day 1, daily gavage of TMZ (653322, Sigma, St. Louis, MO, USA) was performed for the ISO + TMZ group for 21 days, and the ISO group received the same volume of saline. On day 21, the rats were euthanized after subjecting them to echocardiography and the hearts were subsequently harvested. All experiments were approved by the Institutional Research Ethics Committee of Wenzhou Medical University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
7
Isoproterenol-Induced Cardiac Injury and 5-FU Treatment
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Isoproterenol (ISO, Sigma-Aldrich #I6504; St. Louis, MO, USA) was prepared by dissolving a desired amount of powder in NaCl 0.9% [8 (link),9 (link),10 (link)]. The solution obtained was protected from the light and kept on ice until the injections. Before any ISO/Saline injection, the body weight of animals was determined, mice were anesthetized using isoflurane, and baseline echocardiography was obtained. Then, mice were randomly divided in the different groups and, on awakening, prepared to receive saline or ISO at dose of 200 mg/Kg [9 (link),10 (link)]. The solutions were injected subcutaneously under the inter-scapular skin. All ISO injections were administered to male 12/14-weeks-old mice. After 72 h of receiving ISO at dose of 200 mg/Kg, mice received systemic administration of 5-FU (15 mg/Kg/day) for 25 days through subcutaneously implantation of mini-osmotic pumps [9 (link),10 (link)]. Before pumps implantation, the mice were anesthetized using isoflurane. Then, 28 days after ISO injection, 5-FU-realising mini-pumps were removed. The animals were sacrificed after additional 28 days, and the hearts were fixed in 10% buffered formalin or in 4% paraformaldehyde (PFA) for immunohistochemistry analysis or dissociated to obtain a cardiac cell preparation.
8
Isoproterenol-Induced Myocardial Infarction Model
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At 2 months of age, ISO (I6504, Sigma-Aldrich, USA) was administered intraperitoneally 30 mg/kg once a day for 3 consecutive days in both WT and Ucp1−/− rats, while the control group was injected with saline. Rats were randomly assigned to WT-saline, Ucp1−/−-saline, WT-ISO or Ucp1−/−-ISO groups (n = 20/group). After the saline or ISO administration, the four groups of rats mentioned above were performed electrocardiograph (ECG) examination using BIOPAC MP150 physiological signal acquisition system. Briefly, rats were anesthetized with 1.5% isoflurane and placed in supine position. The ECG electrodes were fixed on both upper and right lower limbs of rats and the standard II-lead ECG was recorded. The S-T segment alteration and/or T wave inversion of ECG indicated the successful establishment of ISO-induced AMI models (supplementary material Figure S1).
9
Gefitinib and Isoproterenol Cardiac Tissue
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Ethics statement: All mice (C57Bl6/J, male and female) were purchased from Jackson Labs, housed and underwent drug treatment according to animal care and use protocol #4091, approved by the Temple University Institutional Animal Care and Use Committee, wherein all efforts were made to minimize suffering. The mice were given filter-sterilized i.p. injections of either vehicle (0.1% DMSO [D 4540, Sigma, St. Louis, MO] in PBS) or gefitinib (Gef, 5 mg/kg [G-4408, LC Laboratories, Woburn, MA]) followed 10 min later by an additional i.p. injection of either vehicle (sterile phosphate buffered saline [PBS]) or isoproterenol (ISO, 1 mg/kg [I6504, Sigma, St. Louis, MO]). After either 10 or 60 min the mice were euthanized according to the approved protocol above, their hearts were excised and their left ventricular tissue isolated, snap frozen in liquid N2 and stored at −80°C until further analysis.
10
Quantifying Cardiomyocyte Contractility
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After staining with VSD, cells were qualitatively confirmed to still be spontaneously beating before addition of drugs. Images were captured immediately prior to drug exposure. Medium was then replaced with fresh medium containing either 10−5 M propranolol (SIGMA, P0884) or 10−7 M isoproterenol (SIGMA, I6504). We determined the IC50 of propranolol and EC50 of isoproterenol relative to beat rate for these cells was on the order of 10−6 M and 10−9 M (S1 Fig ). We selected concentrations larger than these to ensure an effective response. Medium without drugs was also replaced as a control. Data was collected 15 min after addition of drugs to ensure complete exposure.
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