The overall goal of this procedure is to examine respiratory control in mitochondria, isolated from biopsied skeletal muscle tissue. This is accomplished by first obtaining a small amount of muscle by percutaneous needle biopsy. The second step is to isolate mitochondria from the biopsied muscle by homogenization.
Next, the isolated mitochondria are washed and separated from the non mitochondrial fractions through centrifugation and passing the sample through a wedded cheesecloth. The final step is to plate isolated mitochondria to perform respiro metric analysis through extracellular flux assays to visualize the oxygen consumption rate. Ultimately, the seahorse extracellular flux analyzer is used to examine respiratory control in isolated mitochondria.
The main advantage of this technique over existing methods is that it requires a minimum amount of muscle tissue as low as 20 milligrams and limits inter experimental variability. These features make this technique suitable for clinical research applications. The implications for this technique are for diagnosing mitochondrial dysfunction in patients.
Visual demonstration of this method is critical as the muscle biopsy and mitochondrial isolation require precise manipulations that are best demonstrated visually rather than solely described in text. To perform the biopsy locally, administer 1%lidocaine, taking care not to infiltrate the muscle. Follow this with a 10 minute wait period.
To permit sufficient numbing, take the biopsies from the middle region of muscle between insertion and origin, avoiding subfascial and myo tendonous areas. To achieve this first, use a percutaneous needle and follow a fascial pop or resistance of the fascia as a guide. Estimate the depth with the anesthesia needle, then feel it with the narrow scalpel blade and make a four to five millimeter incision through the fascia.
Advance the needle through the incision until it is inserted into the muscle. Collect multiple samples with the window turned in different directions. Apply continuous suction using a 60 cubic centimeter syringe while advancing and withdrawing muscle samples into the percutaneous needle two to four times in different directions.
Discontinue the suction and remove the needle. Have an assistant apply firm pressure to the puncture site for five minutes. To establish hemostasis, disconnect the needle from suction tubing and carefully remove the muscle samples from the window and barrel.
Make a second pass if more muscle is needed by repeating this procedure. Remove any visible blood clots from the muscle sample using forceps. Then weigh the sample and immediately place in a tube containing ice.
Cold ECCOs phosphate buffered saline or DPBS. Remove visible connective tissue from the sample using sharp scissors and tweezers.Thoroughly. Wash specimens three to four times with ice cold DPBS buffer.
To remove blood, keep the samples on ice cold DPBS and process as soon as possible. Within 45 minutes of the biopsy, take precaution to carefully remove any tendons or adipose tissue from the muscle sample. Remove a small portion of any extra tissue for storage at negative 80 degrees Celsius.
This may be used for other experiments such as Western blots. Immediately chop the muscle tissue into fine pieces. Using a sterile pair of scissors, then suspend in 500 microliters to one milliliter of a Sistine protein ACE inhibitor, such as nagars dissolved in CP one buffer at a concentration of 0.2 milligrams per gram of tissue.
Mince the sample until it appears well suspended. Follow this with a five minute incubation at room temperature before transferring to ice, making sure that the homogenizer is thoroughly washed.Beforehand. Homogenize the minced tissue treated with nagars.
Using an automated homogenizer. Keep the sample on ice throughout this process. Homogenize each tissue sample four times each time for a pulse of two seconds.
Using the automated homogenizer at a speed setting of 10, 000 RPM, place the sample back on ice. Wash the probe with 70%ethanol followed by distilled water between tissues. Now, wash the homogenized tissue with an equal volume of chapel Perry one or CP one buffer.
Then wash the tissue with twice the volume of CP two buffer. Add the balance tube by bringing water up to the same volume in another conical tube. Collect the content in a centrifuge tube and centrifuge at 600 GS four degrees Celsius or 10 minutes pre-wet the cheesecloth to allow all particles to pass through and not be caught on the cloth.
Pass the supernatant through a wedded cheesecloth, collecting the filtrate and discarding the pellet, thus removing the majority of non mitochondrial fractions. Then ultracentrifuge the supernatant at 10, 000 GS four degrees Celsius for 10 minutes. Suspend the pellet in four milliliters of CP two buffer before centrifuging a second time in the same manner following centrifugation.
Discard the supernatant and resuspend the pate in two milliliters of CP one buffer. At this point, place a small aliquot from this suspension into a separate test tube to be used for protein estimation. Resus, suspend the remaining sample in CP one buffer and centrifuge.
As before, note that the protein plate should be set up with the standards during earlier centrifugation steps. Finally, resuspend the final pellet in a minimal amount of mitochondrial assay solution or mass. Perform XF assays to visualize the oxygen consumption rate or OCR in pico moles of oxygen per minute or absolute levels of oxygen and pH.
In the data output. Add a 10 x concentration of the compounds in one X mass to the ports A through D of the plate based respirometer to give a final concentration as listed in the text protocol. Prepare sufficient volume of compounds for the required number of wells.
Use the protein calculations measured earlier to determine the concentrations of mitochondria to be added. Determine the optimum amount of mitochondria by titrating one microgram, 2.5 micrograms and five micrograms of mitochondria per well. Of the 24 well plate to minimize variability between wells.
First dilute 10 x mitochondria in cold, one x mass plus substrate. Next, deliver 50 microliters of this suspension to each well. Leaving four wells containing only 50 microliters of mass to be used as background readings.
Place the pre soaked calibration plate along with the upper cartridge from earlier into the respirometer to perform the instrument calibration. Preparing for the test plate run centrifuge the plate at 2000 GS for 20 minutes at four degrees Celsius. After centrifugation.
Gently add 450 microliters of one x mass, plus succinate and rotten known to each. Well view the mitochondria under the microscope to ensure homogenous adherence to the well prior to transferring the plate to the XF analyzer.Sequentially. Measure the mitochondrial respiration in real time using the respirometer by programming it using the settings for the respirometer provided in the text protocol shown here are typical Respiro metric profiles driven by complex two using one microgram, 2.5 micrograms, and five micrograms of mitochondria as expected.
Overall, OCR is increased with higher amounts of mitochondria. The calculated respiratory control ratio or RCR for this assay is 7.95, indicating that the mitochondrial preparation is of high quality in order to compare consistency of results when profiling different amounts of mitochondria analysis of variants or Inova was performed and the sum of squares was calculated. Sum of squares or SS is presented for state two, state three, state three, uncoupled antimycin A and RCR for state two and state three measurements one way.
Innova was statistically significant as well as for ANTIMYCIN and RCR. No significant difference was seen for state three uncoupled between groups. These results indicate that five micrograms of mitochondria per well gave the lowest SS compared to other concentrations.
This plot serves as a guide to indicate how much mitochondrial protein can be expected based on the initial muscle sample size. As expected, there is a strong correlation between the amount of muscle processed and the total mitochondrial protein content of the final sample. Once mastered, this technique can be completed in less than three hours if it is performed properly.
Following the muscle biopsy, it is important to make sure that the patient does not take any aspirin or ibuprofen or other over-the-counter non-steroidals for the next three days in order to ensure that there is good healing of the tissues that we have biopsied. The next thing is that we wanna make sure that they don't do any strenuous activity also for the next day and a half, so nothing like jarring. That would be basketball trampoline tillery, but they can do their own normal activities.
We also ask that they keep the area dry for 24 hours, but the first time that it gets wet to be in a shower so that any types of bacteria that may be on the skin that we have not cleaned does not get into the incision. After watching this video, you should have a better understanding of how to biopsy S lateral skeletal muscle tissue, isolate mitochondria, and measure respiratory control.
