CD4+ T cells from wild-type mice were isolated, activated and cultured in the absence or presence of GW0742 as described in the previous paragraph. For certain conditions, 50 μM etomoxir (Sigma) was added as well. After 48 hrs, media (including GW0742, etomoxir, or vehicle) was refreshed and 10 μl/well of a mix of radioactive and non-radioactive palmitate coupled to BSA (2:1 ratio; 15 μM fatty acid-free BSA (Sigma), 30 μM Na-palmitate (Sigma), and 10 μCi (0.83 μM [9,10-3H-palmitic acid (Perkin Elmer)) was added to each well. The radioactive and non-radioactive palmitate was coupled to BSA by first quickly adding the non-radioactive palmitate pre-heated at 70 °C to BSA pre-heated at 50 °C, followed by addition of the radioactive palmitate at this mix at 50 °C. After an additional 24-hr incubation, 100 % trichloroacetic acid (10% final) was added to the cell suspensions and protein was allowed to precipitate. After centrifugation, NaOH (final concentration 0.75 M) was added to the supernatant to increase pH to 12. Subsequently, 400 μl of supernatant was applied to ion-exchange columns (Dowex 1 × 8–200, Sigma), and 3H2O was recovered by eluting with 2.5 ml of H2O. A 0.75-ml aliquot was then used for scintillation counting. Cell number was counted and the results were expressed as CPM × 105/1 × 106 cells.
Etomoxir
Manufactured by Merck Group
Sourced in United States, Germany, France
Etomoxir is a laboratory reagent used in research applications. It functions as an inhibitor of carnitine palmitoyltransferase 1 (CPT1), a key enzyme involved in the mitochondrial beta-oxidation of fatty acids. Etomoxir is utilized by researchers to study metabolic processes and pathways related to lipid metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Oligomycin, by Merck Group (33 mentions)
Rotenone, by Merck Group (28 mentions)
Antimycin a, by Merck Group (19 mentions)
Bptes, by Merck Group (13 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (12 mentions)
Uk5099, by Merck Group (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
2 deoxyglucose 2 dg, by Merck Group (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Oleic acid, by Merck Group (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Palmitic acid, by Merck Group (5 mentions)
Palmitate, by Merck Group (5 mentions)
Antimycin, by Merck Group (5 mentions)
2 deoxy d glucose 2 dg, by Merck Group (5 mentions)
Cerulenin, by Merck Group (4 mentions)
Simvastatin, by Merck Group (4 mentions)
N acetylcysteine, by Merck Group (4 mentions)
Aicar, by Merck Group (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
167 protocols using etomoxir
1
Fatty Acid Oxidation Assay in Activated T Cells
2
Etomoxir's Effects on ATP and NAD(P)H
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To determine ATP effects of etomoxir treatment, tumor cells were seeded in 96-well plates at 5,000–7,000 cells per well and cultured in the presence of 0 or 200 µM etomoxir (Sigma-Aldrich) for 48 h, with triplicate samples for each condition. Relative ATP concentration was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). To determine NAD(P)H effects of etomoxir treatment, HMECMYC-ER cells were seeded in 96-well plates at 2,000 cells per well and cultured in the presence of 0 or 500 nM TAM for 48 h, then 0 or 200 µM etomoxir for 24 h, with six samples for each condition. Relative NAD(P)H concentration was determined using CellTiter-Glo 96 AQueous One Solution Cell Proliferation Assay (Promega).
3
Evaluating Cell Proliferation and Migration
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To evaluate cell proliferation upon different treatments, 1 × 105 cells/well were seeded (t = 0 h) in six well plates and treated with either DMSO, BAPTA-AM (10 μM, Sigma-Aldrich), U-73343 (2.5 µM, Sigma Aldrich), U-73122 (2.5 µM, Sigma-Aldrich), Etomoxir (20 μM, Sigma-Aldrich). 24 h or 48 h later, cells were harvested and counted, normalizing their number to t = 0 h.
For cell migration, MDA-MB-231 CTRL and MDA-MB-231 MCU-KO cells were seeded at low confluency (30%) in 6-well plates. 24 h later they were treated either with DMSO, BAPTA-AM (10 μM, Sigma-Aldrich), U-73343 (2.5 µM, Sigma Aldrich), U-73122 (2.5 µM, Sigma-Aldrich), Etomoxir (20 μM, Sigma-Aldrich) or Oleic acid (500 μM, Sigma Aldrich) in 2% FCS medium. At the same time a linear scratch was obtained on cell monolayers through a vertically held P200 tip. Images were taken 24 h later. “TScratch” software (https://cse-lab.ethz.ch/software/ ) was used for automated image analysis.
For cell migration, MDA-MB-231 CTRL and MDA-MB-231 MCU-KO cells were seeded at low confluency (30%) in 6-well plates. 24 h later they were treated either with DMSO, BAPTA-AM (10 μM, Sigma-Aldrich), U-73343 (2.5 µM, Sigma Aldrich), U-73122 (2.5 µM, Sigma-Aldrich), Etomoxir (20 μM, Sigma-Aldrich) or Oleic acid (500 μM, Sigma Aldrich) in 2% FCS medium. At the same time a linear scratch was obtained on cell monolayers through a vertically held P200 tip. Images were taken 24 h later. “TScratch” software (
4
Cell Cycle Dynamics in Glioblastoma
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To quantify the fractions of actively cycling cells in the population, we employed 2
methods: immunocytochemical labeling of KI67 and FACS-based mitotic profiling. For
KI67 labeling, 13 mm glass coverslips were placed in 24-well plates and coated with
10 µg/mL laminin in Dulbecco's phosphate buffered saline (dPBS) for 2
hours at 37°C. hGBM cells were plated at a density of 10 000 cells per well on
coated glass coverslips for 24 hours in growth medium. Cells were then treated with
10 µL of dPBS, 5 mM etomoxir, or 5 mM linoleic acid for a final concentration
of 100 µM etomoxir (Sigma E1905) and 100 µM linoleic acid (Sigma
L1376). Twenty-four hours after treatment, cells were fixed in 4% paraformaldehyde.
Coverslips with attached cells were subjected to immunohistochemical staining as
described above. In a separate experiment, TdT+ apoptotic cells were
quantified using an enzymatic detection kit as directed (Chemicon S7160).
To perform mitotic profiling, hGBM cells were treated with
dPBS, etomoxir, or linoleic acid. Twenty-four hours after treatment, cells were
labelled with 2 µg/mL Hoechst in permeabilization solution and then subjected
to FACS using LSRII equipment and ModFit software for mitotic profile analysis. For
each experimental assay, each of 3 biological replicates was plated in triplicate for
each treatment group. Groups were compared using 2-tailed t tests in
Excel.
methods: immunocytochemical labeling of KI67 and FACS-based mitotic profiling. For
KI67 labeling, 13 mm glass coverslips were placed in 24-well plates and coated with
10 µg/mL laminin in Dulbecco's phosphate buffered saline (dPBS) for 2
hours at 37°C. hGBM cells were plated at a density of 10 000 cells per well on
coated glass coverslips for 24 hours in growth medium. Cells were then treated with
10 µL of dPBS, 5 mM etomoxir, or 5 mM linoleic acid for a final concentration
of 100 µM etomoxir (Sigma E1905) and 100 µM linoleic acid (Sigma
L1376). Twenty-four hours after treatment, cells were fixed in 4% paraformaldehyde.
Coverslips with attached cells were subjected to immunohistochemical staining as
described above. In a separate experiment, TdT+ apoptotic cells were
quantified using an enzymatic detection kit as directed (Chemicon S7160).
To perform mitotic profiling, hGBM cells were treated with
dPBS, etomoxir, or linoleic acid. Twenty-four hours after treatment, cells were
labelled with 2 µg/mL Hoechst in permeabilization solution and then subjected
to FACS using LSRII equipment and ModFit software for mitotic profile analysis. For
each experimental assay, each of 3 biological replicates was plated in triplicate for
each treatment group. Groups were compared using 2-tailed t tests in
Excel.
5
Etomoxir's Effects on ATP and NAD(P)H
6
Intracerebral Injection of NMO-IgG and Complement
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Mice were anesthetized by isoflurane inhalation or subcutaneous injection of a mixture of 0.79 μg Fentanylcitrate, 25 μg Fluanisone, 1,25 μg methylparahydroxybenzoate and 12.5 μg midazolam in water / 10 g body weight. We observed no differences or grouping in the calculated loss of AQP4 or GFAP with regards to anaesthesia protocol, therefore we conclude that anaesthesia protocols did not affect outcome. Stereotactic coordinates were relative to bregma: 2 mm lateral, 0.2 mm anterior and 3.5 mm ventral. A 30-gauge needle attached to a 50 μl Hamilton syringe was inserted and a total of 6 μl was infused at 1 μl/min (150 μg NMO-IgG (patient-derived) or normal-IgG (healthy donor) + 144 μg complement (C) (healthy donors) (Asgari et al., 2013) (link), with or without etomoxir (0.5 μg, Merck, Sigma-Aldrich, Søborg, Denmark)). Two days later mice received a total volume of 10 μl containing etomoxir (0.5 μg, Merck, Sigma-Aldrich) or vehicle (HBSS) intrathecally via cisterna magna. Mice were sacrificed at day 4. Brains were dissected out and processed as previously described (Asgari et al., 2013) (link).
7
Polarization of Bone Marrow-Derived Macrophages
8
Metabolic Modulation and Cell Viability
9
Cellular Signaling Pathway Modulation
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Dexamethasone (Dex), EVP (NFKB inhibitor), Forskolin, IBMX, db-cAMP, etomoxir, oligomycin, rotenone and CCCP were purchased from Sigma. SP600125 (JNK inhibitor) were purchased from selleckchem and cyclosporine A (CsA) were purchased from Cayman.
10
Antibody Detection of Protein Phosphorylation
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