This method can help answer key questions in the metabolism field such as, how metabolic activity is altered during the process of aging. The main advantage of this technique is the use of an intact body segment which has data that closely resembles the natural and physiological state as compared to the classical mitochondrial isolation. Demonstrating the procedure will be Anuroop and Annika, a grad student in my laboratory.
To begin the protocol, turn on the machine one hour before starting the experiment so that there is adequate time to reach the desired temperature and remain stable. Then, in the software setup, under administration mode, choose the length of the cartridge calibration and the desired temperature. Then, enter the following protocol:Depending on the experimental design, add injection steps from ports A to D after a chosen measurement step.
To check quality and the determination of the basal OCR, adjust the protocol for at least three measurement cycles before setting the injection of the first drug via port A.Pre-calibrate the cartridge one day, or at least four hours prior to testing, by adding 1.0 milliliters of calibrant to each well, and place the sensor cartridge on top of the plate. Cover the cartridge with Parafilm if it is scheduled to hydrate for more than 24 hours to prevent evaporation. Store the cartridge at 37 degrees Celsius without CO2, overnight.
Next, ensure the experimental drugs are thoroughly dissolved in fresh medium, plus 2.5%glucose. Measure and adjust the pH of the drug solution to the pH of the vehicle at the desired temperature. Pipette the drug solution into it's allocated injection port.
Then load the cartridge into the machine and begin calibration. Adjust freshly prepared media plus 2.5%glucose to the desired pH with one normal HCl. Next, prepare an ice box and place a metal plate on the ice.
Open the eyelet plate package. Immerse the nets in media by 16 millimeter Petri dish. Collect one net with the inserter and have the inserter stand up next to the microscope.
Add a small drop of media to the net attached to the inserter. Then, anesthetize the flies by placing them on an ice-cold metal plate. Using forceps, grab the abdomen of a fly and immerse it in the media in a Petri dish under the microscope.
Using a second pair of forceps, gently remove the head of the fly. Place the head in the middle of the net attached to the inserter, and verify that the head is immersed in media. Remove superfluous fluid before centering the heads to avoid losing heads while placing them in the well.
When there are 16 heads on the net, center them. Using the inserter, place the net in the well. Ensure that the heads are trapped under the net and slowly add 700 microliters of media plus 2.5%glucose.
Make sure that the empty wells, which are used for background, also contain a net with 700 microliters of the buffer plus 2.5%glucose. Check the wells for air bubbles under the nets via the microscope. Pipette gently up and down using a one millimeter pipette to remove any bubbles.
Keep the heads centered for a reliable OCR reading. Then add the plate to the machine and start the measurement. At the end of the protocol, remove the cartridge.
Visually confirm that there are no visible leftovers in the port fillings. Next, discard the cartridge and plate if the heads are not to be used for protein extraction. Remember to look at the oxygen and the pH levels, in order to detect any abnormal wells prior to the analysis.
Furthermore, it is important to choose the appropriate oxygen consumption rate calculation, based on the oxygen levels. Upon performing the protocol, it was observed that the oxygen levels of midlife heads dropped quicker than young fly heads. When the range of the oxygen levels is similar between conditions, it is better to use the AKOS algorithm to automatically generate the oxygen consumption rate, or OCR, which reliably mirrors the oxygen level changes during a measurement between young versus midlife heads.
The addition of sodium butyrate, or SB, a KDAC inhibitor, transiently changes the dynamics of the oxygen levels. Whereas the vehicle controls display steady levels of oxygen during the first and last ticks, SB addition causes a considerable and transient drop of the oxygen levels in these ticks. Because the AKOS calculation considers all ticks and ignores an anoxic state, it generates a misleading OCR, as observed in the non-normalized AKOS-based OCR levels, which show little change upon the injection of port A.The fixed algorithm, which more closely models and resembles the OCR and oxygen level changes, reveals an increased OCR upon SB treatment.
When attempting this procedure, it is important to work with two people together in order to ensure a relative short amount of time to prepare a single plate. Following this procedure, other biochemical methods, such as Western Blot and mass spectrometry can be performed to answer questions related to molecular mechanisms, for example, those that moderate metabolic alterations.
