Mitochondrial bioenergetics was measured with the XF24 Flux Analyzer (Agilent Technologies Inc, Santa Clara, CA) as previously described (Krukowski et al., 2017 (link); Ma et al., 2019 (link)). Briefly, tibial nerves were freshly isolated and put into the islet capture XF24 microplate (Seahorse Bioscience, North Billerica, MA) in Seahorse XF base media (#102353-100, Agilent technologies) supplemented with 5 mM glucose (#G7021, Sigma-Aldrich), 0.5 mM sodium pyruvate (#25-000-CI, Corning, Manassas, VA), and 1 mM glutamine (#G8540, Sigma-Aldrich). Oligomycin A (#75351, Sigma-Aldrich, final concentration is 12 μM), Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; #C2920, Sigma-Aldrich , final concentration is 20 μM), and rotenone (#R8875, Sigma-Aldrich, final concentration is 20 μM) and antimycin A (#A8674, Sigma-Aldrich, final concentration is 20 μM) were used to determine mitochondrial respiratory properties. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were normalized to protein contents and were used as indicators for the statuses of mitochondrial oxidative phosphorylation and glycolysis, respectively.
Seahorse xf base media
Manufactured by Agilent Technologies
Seahorse XF base media is a specialized cell culture media designed for use with Agilent's Seahorse XF Analyzers. It is formulated to support the growth and metabolic activity of cells during real-time measurements of oxygen consumption and extracellular acidification.
Automatically generated - may contain errors
Lab products found in correlation
Glutamine, by Merck Group (1 mentions)
Xf24 flux analyzer, by Agilent Technologies (1 mentions)
Xf24 islet capture microplate, by Agilent Technologies (1 mentions)
Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone fccp, by Merck Group (1 mentions)
Oligomycin a, by Merck Group (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
Sodium pyruvate, by Corning (1 mentions)
Xfe24 cell culture microplate, by Agilent Technologies (1 mentions)
Xfe24 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Cb 839, by Selleck Chemicals (1 mentions)
Cb 839, by Merck Group (1 mentions)
Cell mito stress test, by Agilent Technologies (1 mentions)
Xfe96 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Metformin, by Merck Group (1 mentions)
3 protocols using seahorse xf base media
1
Mitochondrial Bioenergetics in Nerve Tissue
2
Mitochondrial Stress Testing of Murine Glioma Cells
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Murine glioma cells (similar low passage N1IC-1/2 and p53-1/2) were seeded in PLL coated XFe24 cell culture microplates (Agilent TEchnologies) at 1.8×104 cells per well in 250μL BFP (n=10 technical replicates for the baseline experiment and n=5 technical replicates for the cysteine/methionine deprivation experiment) and were allowed to attach overnight. For the cysteine/methionine deprivation (CMD) experiment, media was aspirated and replaced with either BFP or CMD BFP media after 4 hours and cells were grown in respective media for an additional 18 hours. Cells were then washed twice in PBS before changing to Seahorse XF base media (Agilent, 102353-100). Mitochondrial stress tests were run with the following concentrations of media: 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate in assay medium, and 2 μM oligomycin, 2 μM trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), and 0.5 μM rotenone/antimycin A. The assay involved injection of glucose (10 mM), followed by oligomycin (1 μM), followed by 50 mM 2-deoxy-D-glucose. Cells were incubated at 37°C in a CO2-free atmosphere for 1 hour. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed using the XFe24 Extracellular Flux Analyzer (Agilent, Santa Clara, CA).
3
Oxygen consumption rate analysis of MG63.3 cells
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Oxygen consumption rates (OCR) were measured using a Seahorse Bioscience XFe96 Extracellular Flux Analyzer. MG63.3 cells were plated at 10,000 cells/well in XF96 cell culture microplates. Following attachment, cells were treated with (1) vehicle, (2) 1 μM CB-839 (Selleck Chemicals), (3) 5 mM metformin (Sigma-Aldrich), or (4) 1 μM CB-839 + 5 mM metformin and incubated for 24 h at 37 °C. Just prior to the Seahorse assay, growth media was replaced with 180 μL of Seahorse XF Base Media (Agilent) supplemented with glucose, glutamine, and sodium pyruvate, and the plate incubated in a 37 °C incubator lacking CO2 for 45 to 60 minutes. OCR was determined by performing the Cell Mito Stress Test (Agilent) according to the manufacturer’s specifications, as previously described [26 (link)].
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