Nrf2 (Active Motif, Carlsbad, CA), antioxidant enzymes and (CAT, GPx, and SOD) and aconitase (Cayman Chemical, Ann Arbor, MI) activities, and other mitochondrial activities (Succinate Dehydrogenase, and NADH Dehydrogenase) (Abcam, Cambridge, MA) were measured using commercially available kits as previously described [32] (link). Nrf2 binding was measured using this kit to best assess its binding to the electrophile response element (EpRE). Nuclear fractions were prepared for Nrf2 activity using a NE-PER Nuclear cytosolic extraction kit (Thermo Fisher Scientific, Waltham, MA). Nuclear purity was cross probed with GAPDH and H3 (Cell Signaling, Danvers, MA). Mitochondrial fractions were obtained according to the aconitase assay instructions. Mitochondrial purity was cross probed using GAPDH and VDAC1 (Abcam, Cambridge, MA). All samples were analyzed in duplicate and run in a single assay with intra-assay and percent coefficients of variability of less than 10% for all assays.
Aconitase
Manufactured by Cayman Chemical
Aconitase is an enzyme that catalyzes the interconversion of citrate and isocitrate in the citric acid cycle. It plays a key role in energy metabolism by facilitating the transformation of citrate to isocitrate, a crucial step in the production of cellular energy.
Automatically generated - may contain errors
3 protocols using aconitase
1
Comprehensive Evaluation of Nrf2 and Mitochondrial Activities
2
Comprehensive Metabolic Enzyme Profiling
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Activities of plasma GGT (BIOO Scientific, Austin, TX), nuclear Nrf2 (Active Motif, Carlsbad, CA), and citric acid cycle and antioxidant enzymes (aconitase, CAT, GPx, SOD, GR, and GST) (Cayman Chemical, Ann Arbor, MI) were measured using commercially available kits as previously described [15] (link). Complex I, Complex II, NADPH ratios, NADH ratios (Abcam, Cambridge, MA), and insulin (Morinaga, Yokohama-Shi, Japan) assays were measured by commercial ELISAs. NADH and NADPH were measured immediately after dissection to avoid sample degradation. All samples were analyzed in duplicate and run in a single assay with intra-assay and percent coefficients of variability of less than 10% for all assays.
3
Mitochondrial and Glycolytic Assessment
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The Seahorse XFe96 Analyzer was used to measure mitochondrial respiration and glycolysis.
Briefly, BMDMs and PerMacs were plated at a density of 5x10 4 cells/well or 2x10 5 cells/ well in 80µL of culture medium in Agilent Seahorse XF96 Cell Culture Microplate. DMF (50 uM) and/or LPS (10 ng/ml) were added 3 hrs after the cells were plated. Following a 16 hr incubation, cells were washed and replaced with XF assay medium (Base Medium Minimal DMEM supplemented with 2 mM Ala-Gln, pH 7.4) prior to analysis. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured after sequential addition of glucose 25 mM, oligomycin (1.5 uM), FCCP (1.5 µM)+ sodium pyruvate (1mM) and antimycin A (833 nM) and rotenone (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
(833 nM). L-Lactate (700510), Aconitase (705502) (Cayman); LDH (MK401) (TaKaRa) assay kits were commercially purchased. Standard protocols provided with the kits were followed when performing all assays.
Briefly, BMDMs and PerMacs were plated at a density of 5x10 4 cells/well or 2x10 5 cells/ well in 80µL of culture medium in Agilent Seahorse XF96 Cell Culture Microplate. DMF (50 uM) and/or LPS (10 ng/ml) were added 3 hrs after the cells were plated. Following a 16 hr incubation, cells were washed and replaced with XF assay medium (Base Medium Minimal DMEM supplemented with 2 mM Ala-Gln, pH 7.4) prior to analysis. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured after sequential addition of glucose 25 mM, oligomycin (1.5 uM), FCCP (1.5 µM)+ sodium pyruvate (1mM) and antimycin A (833 nM) and rotenone (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
(833 nM). L-Lactate (700510), Aconitase (705502) (Cayman); LDH (MK401) (TaKaRa) assay kits were commercially purchased. Standard protocols provided with the kits were followed when performing all assays.
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