从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

The overall goal of this video article is to isolate high quality mitochondria from small quantities of mouse skeletal muscle for use in microplate based tric assays. This method could help answer key questions in biomedical research, such as describing how certain drugs and proteins modulate skeletal muscle, mitochondrial oxygen consumption. The main advantage of this technique is that high quality mitochondria can be isolated from smaller quantities of mouse skeletal muscle than has been previously reported.

Perform euthanasia of the mouse in accordance with institution guidelines. Then position the mouse on its side and use fine tipped scissors to make an incision in the skin. Overlaying the lateral epicondyle of the femur.

Peel the skin towards the mouse, and then remove the fat pad positioned over the quadriceps origin point. Next, cut the quadriceps tendon that is attached to the patella. Slowly snip the apo neurosis between the bone and the quadriceps while avoiding the femoral artery.

Then cut the tendon at the origin point on the femur to liberate the quadriceps muscle and place the quadriceps muscle into chilled PBS. Remove any visible adipose tissue over the quadriceps using a pair of scissors. Once clean, flip the quadriceps over so that the portion of the muscle that was overlying the femur is facing up.

Open the quadriceps muscle with forceps in a fanning motion. Remove the two visible red muscle portions from each lobe and place them into a beaker containing five milliliters of chilled mitochondria. Isolation buffer.

One next, cut the skin overlying the achilles tendon with fine tipped scissors and peel the skin toward the mouse. Cut the exposed achilles tendon and peel the muscle towards the body of the mouse. Cut the tendon at the lateral and medial condyles of the femur to liberate the gastrocnemius and place the gastrocnemius attached to its tendons in a dish filled with chilled PBS.

Flip the gastrocnemius over and peel off the soleus muscle. Place the soleus muscle into the beaker containing five milliliters of chilled mitochondria. Isolation buffer.

One next fan opened the gastroc and removed the three visual red muscle portions. There will be two lateral portions and one medial superficial red strip. Once isolated, transfer the red muscle to the beaker containing five milliliters of chilled mitochondria.

Isolation buffer one. Remove another 75 to 100 milligrams of red muscle from the other leg of the mouse. Repeating the process just shown.

Place the dissected muscle from the second leg into a 1.5 milliliter micro centrifuge tube with the protease and phosphatase inhibitor cocktail and cell lysis buffer prepared as described in the accompanying text protocol. Immediately flash, freeze the second sample in liquid nitrogen for use in western blotting. Place a pre healed flat plastic surface on ice in a large bucket.

Then pour a drop of mitochondria isolation buffer one onto the plastic surface, and use tweezers to place all of the sectioned red muscle into a droplet of isolation.Buffer. Mince the muscle tissue for two minutes using single edge razor blades, change to a new sharp razor every 40 seconds. Transfer the minced tissue to a new beaker with five milliliters of fresh mitochondria.

Isolation buffer one. Then take the solution and drain it through a 100 micron cell strainer, placed onto a 50 milliliter conical tube. Blot the tissue with a delicate task wiper, and then transfer it into five milliliters of a 0.05%tryin solution.

Use tweezers to remove any remaining tissue from the task wiper and add it to the trypsin. Incubate the muscle tissue in the trypsin solution on ice for 30 minutes. Then centrifuge the sample at 200 times G for three minutes at four degrees Celsius.

Following centrifugation, pour the trypsin supinate into a waste container and resuspend the pellet with three milliliters of ice cold mitochondria isolation buffer one. Then transfer the tissue to a 45 milliliter glass homogenizer tube. Rinse the 15 milliliter conical tube with another 1.5 milliliters of mitochondria isolation buffer one to gather any remaining sample and add this to the glass homogenizer tube.

Next, place the glass homogenizer tube into a beaker or plastic container filled halfway with ice. So the glass homogenizer moves minimally within the beaker. Homogenize the sample with a polytetrafluoroethylene pestle attached to a motor driven tissue.

Homogenizer with 10 at 80 RPM. Hold the bottom of each pass for about two seconds. Then transfer the tissue homogenate to a new 15 milliliter conical tube.

And rinse the glass homogenizer tube with 6.5 milliliters of mitochondria isolation buffer one. Pour the buffer into the 15 milliliter conical tube containing the rest of the tissue.Homogenate. Spin the sample at 700 times G for 10 minutes at four degrees Celsius, and then gently pour the supinate into a high strength glass centrifuge tube and discard the pellet.

Next, spin the snat at 8, 000 times G for 10 minutes at four degrees Celsius. To pellet the mitochondria, remove the snat and resuspend the pellet by slowly adding 500 microliters of mitochondria isolation buffer two, cut off the end of a pipette tip and gently homogenize the pellet in the buffer with mixing and stirring motions. Then add another 4.5 milliliters of mitochondria isolation.

Buffer two to the mixture After the pellet is fully suspended, spin the homogenate at 8, 000 times G for 10 minutes at four degrees Celsius to again pellet the mitochondria. Remove the S supernatant and gently but completely resuspend the pellet by adding 2 25 microliter increments of mitochondria isolation. Buffer two, using a pipette tip with its point cut off, gently stir and mix the pellet after each edition of 25 microliters of buffer.

When finished, place this mitochondrial stock on ice. Make two separate one to 20 dilutions of the mitochondrial stock in high purity water. Add 0.1%of Triton 100 x to one sample and sonicate for 10 seconds at a low setting.

Leave the other dilution on ice and return the sonicated sample to ice. After sonication, then perform a citrate synthase assay with both the nons sonicated and sonicated mitochondrial dilution using a photometric assay using standard techniques, determine the percentage of intact mitochondria membranes using the equations provided in the accompanying text protocol. Syndicating the mitochondria results in a statistically significant increase in citrate syntase activity.

Citrate syntase activity in nons sonicated mitochondrial samples compared with sonicated samples shows that 92.5%of mitochondria were intact following the isolation expression of ga. DH and COX four are evident in the whole tissue lysate in the isolated mitochondria expression of Cox four is observed. While only faint bands of GA DH are evident, these findings indicate good mitochondrial isolations with little contamination from non mitochondrial components during the isolation procedure.

While attempting this procedure, it's important to remember to handle the mitochondrial stock with care, never vortexing it or mixing it vigorously following this procedure. Other methods like measuring reactive action species production can be performed in order to answer additional questions like whether an intervention influences reactive oxygen species production in isolated mitochondria.