The respiratory rate was measured using cells resuspended in respiratory buffer [0.5 mM EGTA, 3 mM MgCl2⋅6H2O, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM Hepes, 110 mM sucrose, and BSA (1 mg/ml) (pH 7.1)] and permeabilized by incubation with digitonin (15 μg per 106 cells). The respiratory rates of 3 × 106 to 5 × 106 cells were recorded at 37°C in 2-ml glass chambers using a high-resolution Oxygraph respirometer (Oroboros). Malate (5 mM) and pyruvate (5 mM) were added to provide NADH to complex I. The decarboxylation of pyruvate is catalyzed by pyruvate dehydrogenase (PDH), producing acetyl coenzyme A. Malate dehydrogenase (MDH) located in the mitochondrial matrix induces the oxidation of malate to oxaloacetate. The condensation of oxaloacetate and acetyl coenzyme A induces the formation of citrate by CS. Thus, the addition of malate and pyruvate results in the production of NADH by four enzymes: MDH, PDH, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. Because of the activation of different malate shuttles, complex II is not activated. Citrate and α-ketoglutarate are depleted by antiport transport with malate. Thus, the addition of malate and pyruvate defines the respiration driven by complex I used to compensate the physiological proton leak. Activation of ATP synthesis was induced by the addition of 1.5 mM adenosine 5′-diphosphate (ADP). Thus, we obtained the respiration driven by complex I coupled to the ATP synthesis. The addition of succinate (10 mM) allowed reconstitution of TCA cycle function with the activation of succinate dehydrogenase. Thus, maximal respiration of the mitochondrial chain induced by the competition of the respiration driven by complexes I and II was determined. Addition of rotenone (10 μM) inhibited the electron transfer from complex I to coenzyme Q and allowed the measurement of respiration driven by complex II. Oligomycin supplementation (8 μg/ml) inhibited the ATP synthesis, and respiration uncoupled driven by complex II was determined. Last, FCCP (1 μM) was added to test the quality of the permeabilization of the plasma membrane by digitonin.

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.