Our research focuses on the intersection of metabolism and stress responses in metabolic disease, a major theme of the Washington University NORC program. Through synergisms between our NORC cores, we are able to tackle complex questions in nutritional metabolism and disease through an interdisciplinary and collaborative approach. Traditional mitochondrial respiration assays examined isolated mitochondria.
Isolating mitochondria is time-consuming and can compromise outer mitochondrial membrane integrity. Additionally, mitochondria do not perform alone, but are part of a dynamic network of cellular metabolic adaptation. Our protocol maintains this network to provide a physiologically relevant assessment of mitochondrial function.
Our focus has extended to identifying regulators of metabolic tailoring that are important in health and disease. Key questions we are asking include, Are these regulators, like site-1 protease, equally important across different tissue types? And, What is their physiological significance?
To begin, turn on the high-resolution respirometry and the vacuum system. Remove the stoppers. And then, using a vacuum system, remove the 70%ethanol storage solution.
Refill the chamber with ultrapure molecular grade water. After the final wash, add 2 milliliters of MiRO5 without creatine or blebbistatin to each chamber. Open the respirometry software.
In the pop-up box, set the chamber stir speed to 750 RPM, temperature to 37 degrees Celsius, and data recording interval to two seconds. Set the gain to 1, and polarization voltage to 800 millivolts for the oxygen sensors. Click Connect to Oxygraph to establish communication with the instrument.
In the prompted dialogue box, name and save the experimental file with the current data and calibration. Record oxygen concentration for at least 30 minutes to allow the chambers to warm up and to record signals for air calibration. At the end of the calibration, hold the Shift key and the left mouse button, and drag across a region on the timeline to mark a stable oxygen concentration region.
Navigate to oxygraph followed by oxygen calibration. For air calibration, select the stable oxygen concentration mark and click Calibrate and Copy to Clipboard. Stop the air calibration recording and save the file.
After calibration of the respirometry system, replace MiRO5 from each chamber with fresh 2.1 milliliters of MiRO5 solution. In the respirometry software, enter instrument settings. Set the file name and save settings.
In the next dialogue box, enter the sample information, including the weight of each sample added to each chamber. Now, open the oxygen calibration window. Click Copy from File and select the saved air calibration file.
Then, click the Calibrate and Copy to Clipboard button. Using fine forceps, carefully transfer muscle fiber bundles previously isolated from the mouse into the respiration solution. Place the stoppers on the chamber and push them about halfway to the bottom to semi-close the chamber.
Once the O-rings on the stoppers engage with the chamber wall, use a twisting motion while pushing down to close. When the chamber is halfway closed, a small air bubble is observed at the top of the chamber. Fill a 10 milliliter plastic syringe with pure oxygen from an oxygen tank.
Place the long, blunt needle on the syringe, then insert the needle in the first chamber and slowly deliver approximately 1 milliliter of oxygen. Monitor the chamber oxygen concentration. When the concentration reaches 350 to 400 nanomoles per milliliter, gently twist the stopper while pushing down to fully close the chamber.
Observe the chamber and ensure that no air bubbles remain. To normalize oxygen flux data to the massive tissue from the layout menu, select the layout 6 Specific Flux per Unit Sample layout. Ensure that oxygen concentration and oxygen flux have stabilized following oxygen addition to begin the respiration measurement.
Using a 10 microliter glass syringe, add 2.5 microliters of 0.8 molar malate to each chamber. Press F4 to mark the timeline and label the mark with M.Record stable oxygen flux for one to two minutes. For aerobic glycolytic, add 10 microliters of 2 molar glutamate and 5 microliters of 2 molar pyruvate to each chamber.
Press F4 to mark the timeline and label the mark with GP.Record stable oxygen flux for one to two minutes. Add 10 microliters of 2 molar glutamate and 10 microliters of 10 millimolar palmitoyl-carnitine to each chamber for fatty acid substrates. Press F4 to mark the timeline and label the mark with GPC.
Record stable oxygen flux for one to two minutes. Using a 25 microliter glass syringe, add 20 microliters of 0.5 molar ADP to each chamber. Press F4 to mark the timeline and label the mark with ADP.
Record stable oxygen flux for one to two minutes. Now, add 20 microliters of 1 molar succinate to each chamber. Press F4 to mark the timeline and label the mark with S.Record stable oxygen flux for one to two minutes.
Using a 10 microliter glass syringe, add 5 microliters of 4 millimolar cytochrome c to each chamber. Press F4 to mark the timeline and label the mark with cytochrome c. Record stable oxygen flux for one to two minutes.
Titrate in three 1 microliter boluses of 1 millimolar FCCP using a 10 microliter glass syringe. FCCP addition results in a brief decrease in oxygen flux levels. Wait for the oxygen flux to increase and stabilize before recording.
Press F4 to mark the timeline and label mark with FCCP. Record stable oxygen flux for one to two minutes. Once the assay is complete, gently twist and pull the stopper upward to remove it.
Rinse the chamber thrice with ultrapure water, followed by three times with 70%ethanol. Place the stoppers in the chambers until resistance is felt, but do not close them completely. Then cap the stoppers.
Save the assay file and disconnect the instrument from the software. Finally, turn off the respirometer. In properly prepared mirroring samples, the addition of cytochrome c post ADP did not affect oxygen flux, confirming the integrity of the outer mitochondrial membrane.
However, when tissue samples were not prepared properly, adding cytochrome c post ADP led to a 40%increase in the oxygen flux, indicating damage to the outer mitochondrial membrane.
