The overall goal of this procedure is to isolate skeletal muscle mitochondria by differential centrifugation and determine the respiratory rates of mitochondrial respiratory chain complexes I, II and IV.This method can help answer key questions in the mitochondrial bio-energetic field such as diseases and syndromes associated with mitochondrial dysfunction, such as diverse neurological diseases, sepsis and age-related disorders. The main advantage of this technique is the absence most of the cytologic factors in the isolated mitochondrial preparation, which may interfere with the analysis of the mitochondria functions. Demonstrating the procedure will be Sandra Nansoz, the technician from my laboratory.
To begin this procedure, obtain a beaker of skeletal muscle samples as outlined in the text protocol. Place the beaker on an ice bucket. Using fine scissors, mince the muscle into pieces one to two millimeters in size.
Next, rinse the minced tissue twice with ice cold isolation buffer using 20 milliliters for each wash. Suspend the washed tissue in 10 milliliters of isolation buffer per gram of tissue and then add five milligrams protease-G tissue. Then place the beaker into an ice bath with a magnetic stirrer and stir for 10 minutes.
After this, dilute the suspension with an additional 10 milliliters of isolation buffer supplemented with 0.2%de-fatted BSA per gram of tissue. Next, decant all of the isolation buffer from the beaker. Wash the tissue twice with ice cold isolation buffer supplemented with 0.2%BSA using 20 milliliters for each wash.
Then, re-suspend the tissue in 10 milliliters of BSA supplemented isolation buffer per gram of tissue. Use a semi-automatic glass homogenizer with a loose-fitted pestle to homogenize the tissue. Next, transfer the homogenate to a centrifugation tube and centrifuge at 10, 000 times G for 10 minutes at four degrees Celsius.
Using a serological pipette, discard the supernatant. Re-suspend the pellet with 10 milliliters of ice cold BSA supplemented isolation medium per gram of tissue. Centrifuge the suspension at 350 times G for 10 minutes at four degrees Celsius.
After this, use a serological pipette to reserve the supernatant, discarding the pellet. Filter the supernatant into a beaker kept on ice through two layers of gauze. Transfer the filtered suspension to a centrifuge tube and centrifuge 7, 000 times G for 10 minutes at four degrees Celsius.
Then discard the supernatant. Re-suspend the crude mitochondrial pellet in five milliliters of BSA supplemented isolation buffer per gram of tissue. Centrifuge this suspension 7, 000 times G for 10 minutes at four degrees Celsius.
Next, discard the supernatant and re-suspend the pellet in 20 milliliters of wash buffer. Centrifuge at 7, 000 times G for 10 minutes at four degrees Celsius. Repeat this re-suspension and centrifugation process once more.
After this, discard the supernatant and re-suspend the pellet in one milliliter of wash buffer. Determine the protein concentration using any standard method. Then, keep the concentrated mitochondrial suspension on ice until ready to perform respirometry assays.
To begin, re-suspend the mitochondrial concentrate in the respiration buffer to a final concentration of 0.4 milligrams per milliliter mitochondrial protein. Next, aspirate the respiration medium from the air calibrated oxygraph chamber. Add 2.1 milliliters of isolated mitochondrial suspension.
Insert a stopper to close the oxygraph chamber. Then, stir the mitochondrial suspension continuously at 700 RPM at 37 degrees Celsius. Record the cellular respiration at baseline for three to five minutes until a stable oxygen flux signal is achieved.
To begin complex I-dependent respiration, inject 12.5 microliters of 0.8 molar malate into an oxygraph chamber prepared with the isolated mitochondrial suspension through the stopper's titanium injection port. Then, inject 10 microliters of two molar glutamate. Record cellular respiration for three to five minutes until a stable oxygen flux signal is achieved.
After this, inject 10 microliters of 0.05 molar ADP into the chamber through the injection port. Record cellular respiration until the oxygen flux signal increases then decreases and stabilizes. For the complex II-dependent respiration, inject two microliters of 0.2 millimolar rotenone into an oxygraph chamber prepared with isolated mitochondrial suspension.
Record the cellular respiration until a stable oxygen flux signal is achieved. Then, inject 20 microliters of one molar succinate. Record cellular respiration until a stable oxygen flux signal is achieved.
After this, inject 10 microliters of 0.05 molar ADP into the chamber through the injection port. Record cellular respiration until the oxygen flux signal increases then decreases and stabilizes. For the complex IV-dependent respiration, inject 2.5 microliters of 0.8 millimolar ascorbate into an oxygraph chamber prepared with isolated mitochondrial suspension.
Next, immediately inject 2.5 microliters of 0.2 molar TMPD and 10 microliters of 0.05 molar ADP. Record cellular respiration until a stable oxygen flux signal is achieved. Then, inject 10 microliters of one molar sodium azide.
Record cellular respiration until the oxygen flux signal decreases, then stabilizes. In this study, intact mitochondria are isolated from skeletal muscle. Complex I-dependent respiratory rates are then measured via high resolution respirometry.
As can be seen, the mitochondrial respiration rate increases immediately upon the addition of ADP. The respiration rate decreases after ADP depletion to levels similar to those observed before the addition of ADP. This indicates that the ratio of state 3 and state 4 known as the respiratory control ratio, or RCR, is high and that mitochondrial integrity is well preserved during the isolation procedure.
Complex II-dependent respiratory rates are then observed by inhibiting complex I-respiration with rotenone and injecting the complex II-substrate succinate. ADP is then added, resulting in an immediate increase in respiration. Once ADP is depleted, respiration decreases to levels similar to those observed before the addition of ADP, indicating a high RCR, in that the mitochondrial integrity is well preserved during isolation.
Complex IV-dependent respiration rates are observed by adding ascorbate, TMPD and ADP. Sodium azide is then added to inhibit complex IV.The difference in oxygen consumption before and after the addition of sodium azide is interpreted as the real complex IV respiration. The high complex IV-dependent respiration rate observed in state 3 indicates that mitochondrial integrity is wel preserved during the isolation procedure.
Once mastered, this technique can be done in less than one and a half hours if it is performed properly. While attempting this procedure, it is important to remember to perform a calibration of the oxygen sensors in advance prior to homogenization. Following this procedure, other methods like measurements of cellular ADP content, mitochondrial membrane potential, ATP synthase enzymatic activity as substrate levels can be performed in order to answer additional questions like whether changes in respiratory rates are due to lack of mitochondrial substrate, membrane potential, or reduced ATP synthase enzymatic activity.
After watching this video, you should have a good understanding of how to isolate mitochondria from a skeletal muscle by differential centrifugation and how to measure respiratory rates of mitochondrial complexes I, II and IV using high resolution respirometry. After its development, this technique paved the way for researchers in the field of mitochondrial energy metabolism to explore mitochondria energy function in fields such as genetic mitochondrial diseases, myopathies, ischemia reperfusion injury in isolated mitochondria. Don't forget that working with antimycin A, rotenone, sodium azide and FCCP can be extremely hazardous.
And precautions such as wearing gloves and laboratory coats should always be taken while performing this procedure.
