Oxygen consumption rate was determined using an XF Extracellular Flux Analyzer (Agilent Technologies). MEFs were plated in XF24 microplates (Agilent Technologies) and grown to confluency in Rho 0 media (as described above), and switched to an assay medium composed of Seahorse XF base medium (Agilent Technologies) supplemented with 10 mM glucose (Agilent Technologies), 1 mM pyruvate (Agilent Technologies), and 2 mM l-glutamine (Agilent Technologies), and subjected to the Mito Stress Test Kit (Agilent Technologies) using the standard protocol provided. Briefly, after basal respiration was measured, the MEFs were treated sequentially with 2 μM oligomycin, 2 μM FCCP, and 0.5 μM rotenone. Isolated liver mitochondria (300 μg) were loaded onto XF24 cell culture microplates in KCl buffer containing 7 mM pyruvate and 1 mM malate and subjected to the Mito Stress Test Kit using the standard protocol provided. Briefly, after basal respiration was measured, the mitochondria were treated sequentially with 10 mM ADP, 2 μM oligomycin, 5 μM FCCP, and 0.5 μM rotenone. To interrogate ADP uptake, 200 μg of isolated liver or MEF mitochondria was loaded onto XF24 cell culture microplates in KCl buffer containing 7 mM pyruvate and 1 mM malate and treated sequentially with ascending concentrations of ADP (0.5, 2, and 2.5 mM) followed by 0.5 μM rotenone.

Adenine nucleotide concentrations were measured using chemical assay kits. The Abcam ATP Assay Kit (Abcam) was used to measure mitochondrial ATP levels. Either 2 mg of previously frozen, isolated liver mitochondria or 1 mg of previously frozen, isolated MEF mitochondria was suspended in 100 μl of KCl buffer and diluted with 400 μl of ATP assay buffer. Either 40 μg of liver mitochondria or 20 μg of MEF mitochondria was added to each assay, and ATP levels were measured according to the fluorometric assay protocol. Cell Technology Fluoro ADPTM: fluorescent ADP Cellular and Tissue ADP Detection Kit (Cell Technology) was used to measure mitochondrial ADP levels. Either 2 mg of previously frozen, isolated liver mitochondria or 1 mg of previously frozen, isolated MEF mitochondria was suspended in 100 μl of KCl buffer and diluted with 200 μl of substrate buffer and sonicated for 3 min. One hundred ten micrograms of liver mitochondria or 55 μg of MEF mitochondria was added to each assay, and ADP levels were measured according to the Fluoro ADP Assay protocol (Cell Technology).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.