Peritoneal macrophages were stimulated with MLE-12 or LLC Exo. (40μg/mL) for 16 hours. Cells were then washed and incubated in glucose free RPMI for 30 minutes before 2-NBDG (400μM, BioVision) was added. Cells were incubated with 2-NBDG for 15 minutes before washing and staining with viability dye and F4/80 for analysis via flow cytometry. The following concentrations of metabolic inhibitors or substrates were used for 16-hour cell culture: 2-deoxy-D-glucose (1mM, Sigma), SEITU (2–4mM, Cayman Chemical), Dimethyl-2-oxoglutarate (1mM-5mM, Sigma), Sodium-L-Lactate (20mM, Sigma), AZD3965 (250nM, MedChemExpress). Levels of L-lactate in the supernatants were measured by L-Lactate Assay Kit I (Eton Bioscience, San Diego, CA) according to manufacturer’s instructions.
Azd3965
Manufactured by MedChemExpress
Sourced in Netherlands, Belgium, United States
AZD3965 is a monocarboxylate transporter 1 (MCT1) inhibitor. MCT1 is responsible for the transport of lactate and other monocarboxylates across cell membranes. AZD3965 can inhibit the activity of MCT1, which may have potential applications in research.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Sodium l lactate, by Merck Group (2 mentions)
Sodium lactate, by Merck Group (2 mentions)
Cb 839, by Selleck Chemicals (2 mentions)
U 13c glucose, by Cambridge Isotopes (2 mentions)
U 13c lactate, by Cambridge Isotopes (2 mentions)
L asparagine, by Thermo Fisher Scientific (2 mentions)
L proline methyl ester hydrochloride, by Merck Group (2 mentions)
L glutamic acid dimethyl ester hydrochloride, by Merck Group (2 mentions)
1 13c lactate, by Cambridge Isotopes (2 mentions)
3 4 13c glucose, by Cambridge Isotopes (2 mentions)
L methionine sulfoximine, by Merck Group (2 mentions)
Tgf β1, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Merck Group (2 mentions)
Sodium oxamate, by Cayman Chemical (2 mentions)
L lactate assay kit 1, by Eton Bioscience (1 mentions)
2 nbdg, by Abcam (1 mentions)
2 deoxy d glucose, by Merck Group (1 mentions)
Dimethyl 2 oxoglutarate, by Merck Group (1 mentions)
3 nitropropionic acid, by Merck Group (1 mentions)
7 protocols using azd3965
1
Metabolic Regulation of Peritoneal Macrophages
2
Metabolic Profiling of TGFβ-1 Signaling
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TGFβ−1 was purchased from Peprotech; Amino acids (L-asparagine, L-glutamine, proline), cell-permeable metabolites (L-proline methyl ester hydrochloride, L-glutamic acid dimethyl ester hydrochloride, dimethyl 2-oxoglutarate), sodium lactate and L-methionine sulfoximine were purchased from Sigma; CB839 was purchased from Selleck; AZD3965 was purchased from MedChem Express; sodium oxamate was purchased from Cayman Chemical; stable isotopes ([U-13C] glucose, [3,4-13C] glucose, [U-13C] lactate, [1-13C] lactate) were purchased from Cambridge Isotope Laboratories. An equivalent amount of solvent (DMSO or water) was added to control samples to control for any solvent-based effects.
3
Metabolic Modulators and Inhibitors Protocol
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TTFA (2-thenoyltrifluoroaceton), rotenone, 3-nitropropionic acid, and MCT1 inhibitor (α-cyano-4-hydroxycinnamic acid) were purchased from Sigma-Aldrich. The second MCT1 inhibitor AZD3965 was purchased from MedChemExpress, NL. Stock solutions were prepared in following concentrations; 200 mM TTFA (in DMSO), 80 mM rotenone (in DMSO), 830 mM 3-nitropropionic acid (%70 EtOH-PBS mix), α-cyano-4-hydroxycinnamic acid 200 mM (in 1 M NaOH-PBS mix), AZD3965 100 mM (in DMSO). 2, 2-dichloroacetophenone (DAP) was purchased from Sigma (St. Louis, MO) and stock solutions of the drug were prepared at 7 mM in dimethyl sulfoxide (DMSO). Sodium l -lactate and sodium pyruvate were purchased from Sigma-Aldrich (Cat# L7022 and Cat# S8636, respectively). Quizartinib (AC-220) was obtained from MedChemExpress (Cat# HY-13001).
4
In vivo assessment of AZD3965 effects
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All in vivo experiments were performed with approval (#2019/UCL/MD/027) of UCLouvain Comité d’Ethique pour l’Expérimentation Animale according to national and European animal care regulations. Eight weeks-old male C57BL/6JRj mice (Janvier, Le Genest-Saint-Isle, France) were randomly assigned to an experimental group undergoing sequential tests from the least to the most stressful, as depicted in Figure 7 A. In each group, half of the mice received 100 mg/Kg of AZD3965 (MedChemExpress, Kampenhout, Belgium; Catalogue #HY-12750) per gavage 5 days per week until the end of experiments. The other mice received an equal amount of vehicle (DMSO 0.5%, methylcellulose 0.01%, Tween 80) using the same schedule. All tests are detailed in Appendix A . Mouse weight over 5 weeks of treatment and anal body temperature after 15 days of treatment were also measured. At the end of the experiments, mice were sacrificed by cervical dislocation, and organs (gastrocnemius muscles, whole heart and whole brain) were collected for mRNA and proteins expression analyses.
5
Heterophil Extracellular Traps Induction
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The GT powder was dissolved in DMSO and subsequently diluted with RPMI 1,640 to achieve the desired concentration for experimental use. Following a 20 min incubation of duck heterophils, they were exposed to GT (90, 270, 810 ng/mL) for an additional 1 h of incubation.
Parallel experiments were conducted to investigate the potential mechanism of HETs using the following inhibitors: Diphenyleneiodonium chloride (DPI ) and U0126 (10 μM, MedChemExpress), NSC23766 (200 μM, MedChemExpress), 3PO (24 μM, MedChemExpress), 3-Brouopyruvic acid (3-BP; 3 mM, MedChemExpress), STF-31 (1 μM, MedChemExpress), AZD3965 (1.6 μM, MedChemExpress), and Ritonavir (4.2 μM, Selleckchem). Cells were pre-cultured with inhibitors for 20 min prior to stimulation; zymosan was used as a positive control group. Finally, PicoGreen dye (Invitrogen) was added in equal amounts after incubation and the fluorescence intensity was measured at excitation wavelength of 485 nm and emission wavelength of 535 nm. DNA concentration was determined by conversion based on a standard curve.
Parallel experiments were conducted to investigate the potential mechanism of HETs using the following inhibitors: Diphenyleneiodonium chloride (
6
AZD3965 Inhibitor Evaluates Metabolic Alterations
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Selective MCT1 inhibitor AZD3965 was obtained from MedChemExpress (NJ, USA). Glucose, nigericin, N-acetylcysteine (NAC), RPMI-1640, and antibiotics were purchased from Sigma-Aldrich (Madrid, Spain). Fetal bovine serum (FBS) and C-SNARF-1 AM were both from ThermoFisher (Madrid, Spain). All compounds except Glucose solution, which was dissolved in water, and NAC, which was dissolved in culture media, were dissolved in DMSO and made up with the media so that the final concentration of the vehicle was not > 0.04% (v/v).
7
Metabolic Profiling of TGFβ-1 Signaling
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