The overall goal of this procedure is to isolate mitochondria and perform mitochondrial respiration. This is accomplished by first isolating skeletal muscle from spray dly rats. Next, the muscle is homogenized and the mitochondria are isolated by centrifugation.
The protein concentration is then determined using the Bradford assay. Finally, mitochondrial respiration is measured by polygraphy. Ultimately, results can be obtained that show oxygen consumption through the different mitochondrial respiration states.
This method can help answer key questions in the skeletal muscle field, such as the effect of different interventions of mitochondrial function. Though this method can provide insight into skeletal muscle metabolism, it can also be applied to other organs such as liver, brain, kidney, or almost any tissue Before beginning the procedure. To isolate skeletal muscle, prepare the following solutions, P-B-S-P-B-S plus 10 millimolar EDTA, eight times mitochondria buffer isolation buffer one or IB one, isolation buffer two or IB two and experimental buffer or eb.
See the accompanying written protocol for instructions. Next, turn on the centrifuge and set it to four degrees Celsius. Turn on a water bath and set it to 37 degrees Celsius.
In a nice bucket, put three 10 milliliter beakers, potter elbow, gem tissue grinders, small surgical scissors, and 10 micro centrifuge tubes on ice. Everything must stay ice cold throughout the experiment. Place IB one and IB two on ICE and EB into a warm water bath.
Then add the following solutions to the three beakers on ice beaker one 10 milliliters of PBS beaker, two 10 milliliters of P-B-S-E-D-T-A beaker.Three. Three milliliters of ib. One euthanize a rat according to the procedures approved by your local institutional animal care and juice committee.
Next rapidly isolate 250 to 500 milligrams of skeletal muscle with forceps and mayo Clinic scissors. Rinse it in beca one, then transfer it to beca two, to homogenize the muscle. First, transfer it to beaker three, and finally mince it with scissors.
Transfer the solution to the potter Elva gem homogenizer homogenize the muscle using a motorized pestle 10 times, keeping the tube in ice at all times. Next, transfer the homogenate to pre chilled two milliliter micro centrifuge, tubes and centrifuge at 700 Gs for 10 minutes at four degrees Celsius. Transfer the S supernatant to a new pre-filed micro centrifuge tube and discard the pellet centrifuge.
The S supernatant at 10, 500 Gs for 10 minutes At four degrees Celsius, transfer the SNAT to a new pre-filled micro centrifuge tube and label it with SN one and muscle type Reese. Suspend the pellet in 500 microliters of IB two, then centrifuge at 10, 500 Gs for 10 minutes at four degrees Celsius. Transfer the supinate to a new prefilled micro centrifuge tube and label it with SN two and muscle type.
Suspend the final mitochondrial pellet in 100 microliters of IB two. Spin it in a mini fuge for a few seconds. If there is a pellet, transfer the supinate to a new pre-filled micro centrifuge tube and discard the pellet Calibration of the oxygen electrode is a key step in this protocol and must be performed daily.
Calibration of the electrode can be completed during the mitochondrial isolation. To begin, add 100 milliliters of distilled water to a 250 milliliter flask star vigorously for 20 minutes. To equilibrate the water with atmospheric gas, cover the GRAT electrode with a few drops of 50%potassium chloride.
Next place a small piece of blue rizla rolling paper on top of the electrode. Then a piece of PTFE membrane. On top of the rolling paper.
Apply the inner ring using the ring applicator, and then place the outer ring in the electrode groove. Connect the electrodes and assemble the bigger plastic ring base, mitochondrial chamber and water hoses. Turn on the GRAT boxes and start the GRAS software.
Add the equilibrated water to the mitochondrial chamber. Then add a stir bar to turn on the stir bar. Click on hardware.
Then stir a speed. Adjust the speed to 60 and click on. Next, begin a new experiment.
Click on calibrate and then liquid phase calibration. For box one, change the temperature to 37 degrees Celsius and click okay twice. When override changes to, okay, click it and open the nitrogen gas tank.
Place the tip connected to the nitrogen tank into the chamber and establish zero oxygen. Click okay when it switches from override. Aspirate the water and add 500 microliters of EB to the mitochondrial chambers.
Seal the chamber with a plunger. If using multiple boxes, repeat from the calibration step for each box. Start a new experiment and let it run for about one minute.
To stabilize the signal, add the necessary volume of the mitochondria suspension to arrive at 150 micrograms in each chamber. Mark the event and let it run. For one minute, add 10 microliters of warm 250 millimolar glutamate, 125 millimolar malate for a final concentration of five millimolar glutamate, 2.5 millimolar malate mark, and let it run for one minute.
This is defined as state two. Next, add 7.5 microliters of 10 millimolar a DP for a final concentration of 150 micromolar. Then mark and let it run for 30 seconds.
This is defined as state three. Add another 7.5 microliters of 10 millimolar a DP mark, and let it run for 1.5 minutes. Add 0.5 microliters of cold 10 millimolar oligo mycin for a final concentration of one micromolar mark, and let it run for three minutes.
This is defined as state four. Add one microliter of cold, 0.1 millimolar FCCP for a final concentration of 0.2 micromolar mark, and let RUM for three minutes. This is defined as state five.
When finished, end the experiment and save it. Acquire the respiration rates using the raft software. Enter the normalization factor.
If adding 150 micrograms of mitochondrial protein, the normalization factor will be 0.15. Calculate the normalized respiration rates for the different respiration states. Calculate the respiratory control ratio or RCR by dividing state three by state four.
This figure shows a representative tracing of oxygen consumption by skeletal muscle.Mitochondria. After the electrode signal is stabilized, the mitochondria sample is added to the GRAT chamber. State two, respiration starts with the addition of glutamate and malate.
Addition of A DP increases oxygen consumption. Defining state three. Respiration A TP syntase is blocked by the addition of a liga mycin to obtain state four respiration.
Finally, FCCP is added to uncouple mitochondrial respiration. This diagram shows representative oxygen consumption rates for states 2, 3, 4, and five. The respiratory control ratio or RCR is calculated for each experiment.
An RCR greater than four is considered evidence of a viable mitochondria preparation While attempting this procedure. It's important to keep mitochondrial samples in ice at all times. Following this procedure.
Other methods like Western blots can be performed in order to answer additional questions like content of mitochondrial proteins. After watching this video, you should have a good understanding of how to isolate mitochondrial from skelet muscle as well as mitochondrial respiration.
