This protocol is significant because it allows users to prepare mitochondria samples from frozen tissues collected from previous studies and archived. This protocol also reduces the use of live animals for tissue harvest. This technique allows users to use previously frozen archive tissues to prepare mitochondria enriched samples by employing a gentle tissue shredding method.
This method of preparation maximizes the yield of mitochondria preparation. Through reading of the protocol is advisable to ensure the availability of all components and tools especially the fully charged shredder drive and also exercise with some unwanted tissues to get familiar with the steps. Demonstrating the procedure will be Edina Kosa, a Research Associate from my laboratory.
To begin with, record the weight of the tube containing the heart tissue. Now weigh the empty tube and subtract it from the previously recorded weight to obtain a net weight of the heart tissue. Put the frozen heart tissue directly into the beaker of ice-cold solution.
Stir it for five minutes. Simultaneously using tweezers apply slight pressure to the tissue to get out as much blood as possible. Take the tissue out with forceps and squeeze the heart with a clean filter paper towel to remove blood.
Then place the tissue into another beaker of ice-cold solution. After stirring the tissue for five minutes, gently dry the tissue on a paper towel and remove the fat, clotted blood, auricles and fasciae. Pool the ventricular tissue.
To shred the tissue samples, place 50 to 300 milligrams of heart tissue into the tissue shredder chamber from the ram side and close the ram side. Then add about 0.2 to 0.8 milliliters of washing buffer to the cap side of the tube. Use the tube tool to cap the shredder chamber with the shredder cap snugly without over tightening it.
Place the shredder chamber into the shredder holder unit with the ram side down for engaged fitting. Insert and engage the bit of the fully charged shredder driver with the shredder cap ensuring the bit is snugly locked into place then apply pressure down to shredder driver in shredder chamber until the pressure indicator on the shredder holder reaches a setting of approximately two. Press the trigger and run the shredder driver for about 10 to 12 seconds while maintaining the setting at two.
Observe the tube to ensure shredding and passing of all or most of the tissue mass through the lysis disk into the upper shredder chamber and if necessary return the tube to the shredder holder to continue shredding. After shredding the sample lift the shredder chamber out of the holder unit and use the tube tool to remove the shredder cap and place it on a clean surface for later use recommended only within the same sample to avoid cross contamination from other tissue samples. Transfer the shredded heart tissue into the third beaker with 40 milliliters of washing buffer with a stir bar and stirred the tissue for five minutes at a medium speed while on the stir plate.
Filter the suspension through a pre-wetted nylon mesh monofilament. After discarding the filtrate wash the shredded tissue on the filter three times with 10 milliliters of washing buffer. Transfer the washed tissue into a 50 milliliter beaker with 20 milliliters of washing buffer placed in the ice bath on the magnetic stir.
Add 0.5 milliliter of trypsin solution while constantly stirring for 15 minutes. Approximately halfway through the stirring transfer the tissue suspension into a loosely fitted handheld glass-on-glass homogenizer that can hold 15 milliliters of volume. Homogenize the suspension with four to five gentle strokes without producing froth.
Then, place the homogenate into a 50 milliliter beaker and mix on a stirring plate. Next, add 10 milliliters of the isolation buffer containing trypsin inhibitor to the homogenate and incubate while gently stirring. After filtering the suspension again through a nylon filter save the filter in a 50 milliliter conical tube and transfer the particulate fraction left on the filter to the glass-on-glass homogenizer.
At 15 to 20 milliliters of the washing buffer and gently homogenize with the hand for 25 to 30 seconds. After combining the homogenate with the filtrate and balancing the tubes, centrifuge and then using a plastic pipette remove the supernatant leaving 0.2 milliliters from the top of the fluffy pellet. Filter the resulting supernatant into a new conical tube using an nylon filter to avoid contamination and centrifuge one more time.
After discarding the supernatant wash the pellet two to three times by adding the isolation buffer to the side of the tube to prevent breaking or contaminating the pellet. Discard the fluffy white outer rim layer each time. Add five milliliters of the isolation buffer to each tube and break up the pellet using a pipette with a one milliliter tip or a new transfer pipette Then dilute the suspension with 20 milliliters of additional isolation buffer and centrifuge the suspension at 8, 500 X g for 15 minutes at four degrees Celsius.
After discarding the supernatant resuspend the pellet firstly in five milliliters and then in an additional 15 milliliters of the resuspending buffer and centrifuge at 8, 500 X g for 15 minutes at four degrees Celsius. Now discard the supernatant and save the brown pellet containing washed intact mitochondria. Resuspend the mitochondria enriched pellet in 250 microliters of the resuspending buffer including protease inhibitors.
Aliquot the suspension in 25 microliters volume, quick freeze in liquid nitrogen and store in a 80 degree Celsius deep freezer for several years years. Following the protocol the mitochondria-enriched protein was prepared from the heart of offspring born to two groups of sows. One was exercised during pregnancy and the other was sedentary.
ETC protein profile analysis revealed that the offspring of exercised sows presented relatively reduced levels in some of the ETC complexes. Mitochondrial proteins were resolved in a 3 to 8%gradient BN-PAGE and immunoprobed with antibody cocktails. Both exercised and sedentary groups exhibit multiple supercomplexes.
The prominent supercomplex increase observed was at nine months in the exercised group compared to the sedentary group. Mitochondria-enriched proteins obtained from different age groups of 48 hours three months, six months and nine months were analyzed for the Complex I-II activity using a kinetic assay. The exercised group showed an Complex I-II activity as compared to that of the sedentary group.
Feasibility of functional studies on mitochondria obtained from frozen tissues and cells is shown by other investigators. The preparation of mitochondria obtained from frozen tissue may be applied for a wide variety of tissues with minimal damage.
