After calibration of the respirometry system, replace MiR05 from each chamber with fresh 2.1 milliliters of MiR05 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 one 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 mass of tissue from the layout menu, select the layout 06 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 two molar glutamate and five microliters of two 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 palmitoylcarnitine 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 one 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 five microliters of four 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 3 one microliter boluses of one millimolar FCCP, using a 10 microliter glass syringe.
FCCP edition 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 murine 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.
