This word describes respiration analysis in processing mitochondria from mouse skeletal muscle. This protocol considerably reduce purification time and allows multiple samples to be processed simultaneously. Mitochondria from animals at any eight gender of United background can be mutilated using this protocol enabling the study of respiration in a wide variety of experimental conditions.
This method is suitable for all research areas where mitochondria function is relevant including aging, drug testing, cancer, or development. It can even be adapted to other tissues and organisms as well. Ensure all materials are cold by placing everything on the ice during the procedure to protect mitochondria from damage.
Place three 50 milliliter beakers per sample on ice and add 10 milliliters of PBS in beaker one, 10 milliliters of 10 millimolar EDTA PBS in beaker two, and four milliliters IB1 in beaker three. Spray the right hind limb of a euthanized mouse with 70%ethanol to prevent fur from shedding and tape the limb to the cork dissection board then tape the left fore limb. Make an incision with a sterile disposable scalpel through the skin from knee to toe.
Grip the skin with toothed forceps at ankle level and cut it with fine scissors around the ankle. Ease the skin away from the underlying musculature with fine tip tweezers in one hand while pulling it up with toothed forceps in the other hand. Dissect all skeletal muscles from ankle to knee and remove all connective tissue over the tibialis anterior muscle using fine tweezers and scissors.
Find the four distal tendons of the extensor digitorum longus muscle and section them close to their insertions in the toes. Locate the tibialis anterior distal tendon and cut it close to its insertion below the ankle. Pull the tibialis anterior and extensor digitorum longus tendons above the ankle to liberate the loose ends.
Grip the loose ends of the tendons and ease the muscles away from the rest of the musculature in bones by pulling them up. Cut the proximal tendons of these muscles as close as possible to the kneecap. Turn the mouse upside down to proceed with gastrocnemius and soleus muscle extraction.
Grip the pocket formed between the biceps, femoris, and the gastrocnemius and separate these muscles using fine scissors to visualize the proximal gastrocnemius tendon. Grip and carefully cut the distal Achilles tendon with fine scissors. Cut the proximal gastrocnemius tendon liberating it with the underlying soleus.
Carefully dissect the remaining muscles and re-pin the mouse in the initial position to dissect the quadriceps muscle. Discard the adipose tissue then insert fine tweezers between the quadriceps and the femur to separate the muscle from the bone. Grip the distal quadriceps muscle tendons and cut the tendon as close as possible to the kneecap.
Pull the quadriceps up and liberate them with fine scissors at the proximal insertion. Repeat the same for the left hind limb. Rinse all the muscles in beaker one, then two, followed by beaker three.
Finally, mince all the muscles in beaker three with sharp scissors on ice. Transfer the suspension to a C tube, tightly close it, and attach it upside down onto the sleeve of the homogenizer. Divide the homogenate into two 2 milliliter pre-chilled micro centrifuge tubes and centrifuge for 10 minutes.
Then transfer the supernatant to new pre-chilled tubes and centrifuge for another 10 minutes. Combine the pellet in 500 microliters of IB2 Transfer the supernatant to a new 2 milliliter pre-chilled micro centrifuge tube and label it as supernatant number two, or SN2. Re suspend the final mitochondrial pellet in 200 microliters of re suspension buffer.
Quickly set aside 10 microliters for protein quantification and immediately add 10 microliters of 10%FFA BSA to the remaining mitochondrial suspension to prevent damage. Centrifuge the concentrated mitochondrial suspension at 10, 500 times G for 10 minutes at four degrees Celsius. Re suspend the mitochondrial pellet in 100 microliters of 1X coupling assay medium with BSA, 1X electron flow assay medium with BSA, or 1X beta oxidation medium with BSA, depending on the protocol to be performed.
Dilute this concentrated mitochondrial sample to 0.2 micrograms per microliter using the corresponding assay medium. Seed 50 microliters of the suspension per well in a pre-chilled 24 well micro plate on ice. In the background correction wells, add the corresponding assay medium only.
Store the remaining mitochondrial suspension at minus 80 degrees Celsius for fragmentation purity determination. Spin the micro plate in the pre chilled preparative centrifuge for 20 minutes. In the meantime, warm the remaining assay medium to 37 degrees Celsius.
After centrifugation, leave the micro plate on the bench for five minutes to equilibrate. Add 450 microliters of the warm assay medium per well slowly and carefully to avoid detaching of mitochondria. Place the micro plate immediately in the oxygen consumption measuring instrument without the lid and start the measurement program.
Ensure that the first step is a 10 minute incubation to allow the micro plate to warm up. Once the experiment is finished, remove the cartridge and micro plate, switch off the instrument, and start the analysis. General protein profiles from the different fractions obtained through serial centrifugations showed distinct protein populations in the isolated fractions.
The presence of all the mitochondrial content is evident by the strong signals for markers of the outer and inner mitochondrial membrane and nucleoid associated proteins. Fraction N represents nuclei and undisrupted material. Most of the endoplasmic reticulum, plasma membrane, or cytoplasm markers in the SN1 or SN2 fractions highlight the high purity obtained during isolation.
An increase in oxygen consumption was observed after ADP addition in the coupling and beta oxidation assays. After carbonyl cyanide-p-trifluoromethoxy phenylhydrazone addition, oxygen consumption reached its highest level. A low respiratory control ratio ensures the integrity of the isolated mitochondria.
The electron flow assay examined the activity of the electron transport chain complexes individually or in combination through the injection of specific substrates and inhibitors. Isolating mitochondria are extremely sensitive. Hence all the steps after tissue mitigation need to be performed diligently and carefully to preserve their viability.
Experimental assays can be complimented with a deterministic sematic analysis of key mitochondria molecules or with the study of the assembly of respirometric complexes using blue native polyacrylamide gel electrophoresis.
