The development of a mobile mitochondrial laboratory allows physiologists to take their work into the field, and as a result, they can evaluate a fascinating diversity of energetic challenges that animals display. Captivity is stressful for many animals, so with a mobile lab, we can now take the lab to the animals rather than bring the animals back to the laboratory. With the ability to isolate and measure mitochondrial respiration in the field, techniques that were primarily used by biomedicine can now be used in ecological and evolutionary context.
Begin by parking the mobile laboratory on flat ground, then turn on the generator, and level the vehicle. Extend the slide before setting up the equipment. Proceed to set up and calibrate the mitochondrial respiration chambers to the desired temperature of experiments and the current barometric pressure as per the manufacturer's instructions.
Use a cut five milliliter pipette tip to transfer the minced tissue to a 50 milliliter centrifuge tube, and homogenize the tissue with a blade at 50%power for five seconds. To digest the tissue, add five milligrams of protease per gram of the wet muscle for seven minutes, mixing the solution every 30 seconds. Terminate the reaction by adding an equal volume of the isolation buffer containing BSA.
Next, centrifuge the homogenate at 500 G for 10 minutes, then transfer the supernatant using a cut five milliliter pipette tip through the double layered cheesecloth into a clean 50 milliliter centrifuge tube, then centrifuge the filtered supernatant at 3, 500 G for 10 minutes to precipitate a brown mitochondrial pellet. After discarding the supernatant, add the same volume of the isolation buffer containing BSA to the centrifuge tube, and resuspend the mitochondrial pellet with a flexible scraper by gently working the mitochondrial pellet off the walls of the centrifuge tube, then centrifuge the tube at 3, 500 G for 10 minutes. Resuspend the pellet in the isolation buffer without BSA to repeat the centrifugation step as described earlier.
After the second centrifugation, resuspend the mitochondrial pellet in the resuspension buffer with a clean, flexible scraper, and transfer the resuspended mitochondria to a Dounce homogenizer with a cut one milliliter pipette tip. With the homogenizer, carefully homogenize the mitochondrial suspension with four to five passes. Use a fresh cut one milliliter pipette tip to transfer the homogenized mitochondrial suspension in a labeled two milliliter microcentrifuge tube.
For the mitochondrial respiration measurements of the complex one substrates, add 945 microliters of the respiration buffer to the mitochondrial respiration chamber. Ensure that the stirrer is spinning when the buffer temperature is maintained at 37 degrees Celsius. Seal the chamber with the top, and then start recording the data collection.
After the oxygen concentration has stabilized, add 20 microliters of the mitochondrial suspension to the chamber, and place the lid. In the software, denote that the mitochondria were added to the chamber. Add 10 microliters each of one molar glutamate, 200 millimolar malate, and 200 millimolar pyruvate to the chamber with the individual syringes, and wait until the signal stabilizes before indicating the added substrates in the software.
Use a separate syringe to add five microliters of adenosine diphosphate, or ADP, to the chamber. Denote the added ADP in the software, and observe the rapid oxygen consumption of state three. After state three respiration and mitochondrial oxygen consumption has slowed for four minutes to indicate state four respiration, terminate the recording and save the data file.
For the complex two substrates, repeat the procedure by adding 963 microliters of the respiration buffer to the chamber. After adding 20 microliters of the mitochondrial suspension, sequentially add two microliters of four micrograms per microliter rotenone and 10 microliters of 500 millimolar succinate to the chamber using the separate syringes. When the signal stabilizes, denote the substrate addition in the software.
With the addition of five microliters of ADP, observe the rapid oxygen consumption of state three and indicate the addition of ADP in the software. After state three respiration and mitochondrial oxygen consumption has slowed for four minutes to indicate state four respiration, terminate the recording and save the data file. The representative results indicate the raw mitochondrial respiration data.
The steep slope of step three in the complex one substrates represents the high maximal respiration rate. The successful isolation of the mitochondria from the hind limb skeletal muscle was observed by the sharp turn and the stabilization of a new slope of state four. A similar pattern could be observed for complex two driven mitochondrial respiration.
The poor functioning of the mitochondria resulted in the coupling of the mitochondria for the complex one driven mitochondrial respiration. However, another typical example of poor mitochondria functioning showed the uncoupled complex two mitochondrial respiration with a flat line after the addition of ADP, and no turn to produce the state four data. The numerical values of state three, state four, and the respiratory control ratio, or RCR, for both complex one and complex two were determined from the respiration measurement data During collection, additional tissues can be flash frozen in order to use them in the future for measuring mitochondrial density or oxidative damage.
In addition, after the procedure, isolated mitochondria can also be frozen to measure the activities of electron transport and chain complexes. The capacity to isolate and measure mitochondrial respiration in the field will pave the way to improve our understanding of the role of mitochondria in the evolution of complex life.
