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 two milliliters of MiRO5 without creatine or blebbistatin to each chamber. Open the respirometry software. In the pop-up box, set the chamber stirrer speed to 750 rpm, temperature to 37 degrees Celsius, and data recording interval to two seconds.
Set the gain to one 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.
