The overall goal of this procedure is to assess membrane repair capacity in both the animal and isolated cells. Isolated skeletal muscle fibers are collected and surrounded with media containing FM 1 43 dye. A UV laser is then used to irradiate and damage a portion of the cell membrane to allow entry of the dye into the cell.
Isolated cells are transfected with plasma DNA expressing GFP MG 53, which will localize to the plasma membrane and cytosol of the cell. The tip of a micro electrode is then inserted into the C two C 12 membrane to induce damage. Ultimately, results can be obtained that show translocation of the GFP MG 53 to the injury Site.
Visual demonstration of this method is critical since practices required to master the techniques of micro electrode penetration and muscle fiber isolation. Demonstrating the procedure will be Dr.Lin, our lab manager, Dr.Wazu, a postdoc fellow Matt Orange, a graduate student, and Dr.Shaza, a research assistant professor in our department. To begin place a tray or a blue lab pad underneath the treadmill to collect waste from the animals during the experiment.
Next, set the angle of the treadmill surface. Some treadmills have an integral apparatus to adjust the incline while others require the treadmill to be elevated through other means. A flat surface or an angle between seven and 15 degrees downhill or uphill is generally used.
Acclimate the mice by placing them onto the treadmill with the electric grid off and the belt drive motor on, but not moving for 15 minutes after the acclimation period, turn on the motivational electric grid. Start the treadmill with an initial speed of approximately five meters per minute. With speeds increasing by one to two meters per minute each minute allow the animal a five minute warmup period.
Repeat this warmup period every day for three to 10 days to increase compliance during testing. To run an acute exhaustion exercise. Increase the speed of the treadmill over time until a maximum speed is achieved and monitor for signs of exhaustion.
An animal is considered exhausted when it spends more than half of the time off the treadmill or five consecutive seconds on the grid. Remove the exhausted mouse and record the total running time. Endurance training.
Running protocols start with the same warmup procedure. However, the maximum speed is generally lower and the running times can be quite long. Return mice to their home cage after testing and clean the treadmill with 70%ethanol.
Isolate the superficial layer of the flexor digitorum brevis muscle from the foot for further analysis. First, cut the skin of the sole at the median line, taking care not to damage the muscle underneath. Next, make horizontal cuts and remove the skin.
Identify the flat white tendon of the FDB muscle attached to the calcaneus bone. Use forceps to separate and then sever the tendon close to the site of attachment. Pull the tendon up gently while cutting away any connective tissue.
Locate where the distal tendons branch into the individual digits. Cut the tendons where they connect to the deeper layer of muscle and remove the FDB. Place the FDB muscle bundle into one milliliter of collagenase solution Prewarm to 37 degrees Celsius.
Tape the tube horizontally onto a 37 degree Celsius orbital shaker and shake at 200 RPM for 65 minutes. Sufficient digestion is marked by afraid appearance and pale color. Cut the end of a one milliliter pipette tip to allow the digested bundle to pass through.
Easily transfer the bundle into a centrifuge tube containing about 600 microliters of 2.5. Calcium tyro. Gently flip the tube to shake the loose fibers from the bundle.
Next, cut the end of a 30 microliter pipette tip so that the diameter is only slightly smaller than the muscle bundle. Use this tip to draw the bundle into the pipette and then push it back into the solution. Repeat this process until the majority of the fibers are disassociated from the bundle.
Mix the disassociated fibers by gently turning the tube. Remove the desired amount and drop onto a glass bottom delta T dish. Store the remaining fibers at four degrees Celsius for approximately six hours for use and additional studies.
Allow the dish to sit undisturbed for five minutes. This allows the fibers to adhere to the glass bottom of the dish. Place the glass bottom dish on a confocal microscope equipped with a UV laser.
Observe the fibers under phase microscopy at greater than 100 x magnification to check for the presence of a normal striation pattern and a straight rod like shape. Add FM 1 43 or FM 4 64 styro perineum dies to solution to a final concentration of 2.5 micromolar. Orient the fiber at a 45 degree angle from the top left of the field of view to the bottom right to induce damage.
Irradiate a five by five pixel area of the plasma membrane. Using a UV laser set to maximum power for five seconds, the irradiated region should split the plasma membrane that is half of the five by five box should be inside the fiber while the remainder should be outside the fiber capture images of dye entry into the fiber at intervals of five seconds each for up to five minutes. Only three fibers may be irradiated per dish as the fluorescent dye will eventually be endocytose into the fiber and complicate any data analysis.
After three fibers are used, a new dish should be prepared. Using analysis software, calculate the change in fluorescence intensity in an area approximately 200 square microns for comparison between fibers. The following calculation should be made for each frame delta F divided by F zero, where F zero is the mean fluorescence of the region of interest in the first captured frame and delta F is the change of fluorescence in each subsequent frame.
Use standard methods to transfect cells with plasmid DNA expressing GFP MG 53. Then prepare the micro pipettes. Place a Pyrex capillary on a micro pipette.
Pull and pull by preset program. Attach the micro pipette to a three axis micro manipulator culture. Media is removed from the delta T dish and replaced with 2.5 millimolar calcium tyro buffer.
Place the dish containing transfected cells onto a laser scanning confocal microscope with 40 x 1.3 NA oil immersion objective. Induce acute live cell damage by inserting the micro pipette into the cell membrane and then quickly retracting the micro pipette out of the cell. Collect consecutive live cell images at an interval of 1.54 seconds per frame for the troponin induced membrane disruption.
PERFU 0.005%troponin at a rate of approximately one milliliter per minute directly above the cell being imaged. The treadmill can be used to measure a reduction in running capacity. The MG 53 knockout mice seen here have reduced running capacity due to damage of the skeletal muscle.
The extent of damage caused by the UV laser depends on the membrane repair capacity of the fiber seen. Here is a fiber prior to injury in a normal mouse. This image was captured 200 seconds later and shows a slight injury.
A muscle fiber derived from the MG 53 knockout mice with compromised membrane repair capacity will display more significant dye entry after injury seen. Here is an example of an undamaged GFP MG 53 transfected C two C 12 myo tube. The arrow in this image points towards GFP MG 53 containing vesicles that move towards the sites of damage.
In this example, saponin was used to damage the plasma membrane. Notice the translocation of GFP MG 53 from cytosol to the cell membrane after injury. While attempting this procedure, it's important to remember to isolate good muscle fibers with smooth saral membranes and clear striation patterns.
After watching this video, you should have a good understanding of how to assess the membrane repair capacity of muscle and the culture, the cells.
