To isolate mitochondria, place the anesthetized decapitated rat in a prone position on ice. After exposing the heart, cut the aorta approximately four to six millimeters above the aortic root and below the carotid branching points. Cannulate the aorta and perfuse the heart with ice cold cardioplegia solution using a gravity dependent pressure head until the coronary arteries are cleared of blood and the organ appears blanched.
Dissect away the atria, cartilaginous valvular tissue and fatty tissues to isolate the ventricular myocardium. Mince the tissue with sharp surgical scissors until the pieces are approximately one cubic millimeter. Transfer the minced tissue into the pre-chilled centrifuge tube, labeled spins one and two containing the protease solution.
Add ice cold isolation buffer to a final volume of 25 milliliters. Using a motorized handheld rotor stator homogenizer, disperse the tissue at 18, 000 RPM on ice for 20 to 25 seconds. Centrifuge the homogenized tissue at 8, 000 G for 10 minutes at four degrees Celsius.
Discard the supernatant. And gently rinse the pellet with five milliliters of isolation buffer to remove residual protease. After discarding the rinse, add ice cold isolation buffer to a final volume of 25 milliliters.
Then vortex to resuspend the pellet. Centrifuge the tissue homogenate at 800 G for 10 minutes at four degrees Celsius. Gently pour the supernatant containing mitochondria into a labeled pre chilled 50 milliliter tube.
Now centrifuge the supernatant at 8, 000 G for 10 minutes at four degrees Celsius. Discard the supernatant and retain the mitochondria containing pellet. Using a lint-free wipe, absorb excess supernatant from the inside wall of the tube without disturbing the pellet and place the tube on ice.
Add 80 microliters of ice cold isolation buffer to the bottom of the tube and gently resususpend the mitochondria using a micro-pipette. Once resuspended, transfer the mitochondria to a pre-chilled labeled micro centrifuge tube. Determine the mitochondrial protein concentration using the bison kinetic acid protein assay.
In a pre-chilled micro centrifuge tube, dilute the mitochondrial stock to the desired working concentration with an isolation buffer. To test the viability and quality of mitochondrial isolates, load 2.3 milliliters of respiration buffer into Oxygraph chambers. Allow the oxygen consumption signal to equilibrate at 37 degrees Celsius for about 10 minutes or until the rate is near zero.
Upon equilibration, push down the stoppers and aspirate the excess buffer. Using a 10 microliter Hamilton syringe, add one millimolar EGTA to chelate any residual calcium in the respiration buffer on the Oxygraph. To fuel respiration, add five millimolar sodium pyruvate, and one millimolar al-malate into the buffer.
Then add a bolus of diluted mitochondria and allow respiration to occur for five minutes. At the five minute mark, add a bolus of 500 micromolar ADP to initiate state three respiration. To calculate the respiratory control ratio, divide the maximal rate of oxygen consumption during state three by the rate of oxygen consumption just before the addition of ADP in state two.
A rapid increase in oxygen consumption was observed immediately after ADP addition indicating successful initiation of oxidative phosphorylation in isolated cardiac mitochondria. Heart mitochondria from guinea pigs, Sprague-Dawley rats and Friend leukemia virus B mice, demonstrated high respiratory control ratios, indicating good mitochondrial isolation quality. Liver and kidney mitochondria from Sprague-Dawley Rats also exhibited acceptable respiratory control ratios supporting successful isolation.
HEK293 cells exhibited respiratory control ratio values above the acceptable threshold, confirming the quality of mitochondrial isolation from these cells.
