Hi, I'm Juwan Ma from the Department of Anesthesia at Bergham Women's Hospital in Boston. Today we will show you a technique for acquiring in vivo fluorescence images to monitor the progress of muscle regeneration in mice. We use this procedure in our lab to study the engraftment of transplanted muscle stem cells.
So let's get started. GFP labeled cells for transplantation are isolated and cultured from GFP transgenic mice Before continuing a toe pinch confirms the mouse is fully anesthetized. The hind leg muscles are then removed Under aseptic conditions, the muscle is then macerated.
Satellite cells are dissociated by incubating the mince with a 2%dis displays collagenase solution at 37 degrees Celsius for one hour. After the incubation growth medium containing ham F 10 and 20%FBS is added to stop enzymatic digestion. The muscles then dissociated through repeated tri rating with a pasta perpe.
Next cells are filtered through a cell strainer and then plated onto an uncoated cultured dish. The dish is placed in a standard cell culture incubator for one hour. In order to separate satellite cells from other cell populations with stronger adhesion properties, the supernatant is decanted onto a collagen coated dish, an incubated for another hour.
After this incubation, the resultant satellite cell enriched supinate is removed once again and applied to a new collagen coated plate and cultured until myoblast colonies are observed. This process is termed prepl cells are passaged upon reaching 60 to 70%cellular confluence. A small proportion of cells are used for immuno cyto chemistry, staining with myogenic cell markers to verify the purity of muscle cells in the culture.
Once the purity of myogenic cells is confirmed, the cells are expanded in the same culture. Conditions of transplantation prior to the cell transplantation cells are detached with N 0.25%Trypsin. EDTA cells are twice washed and resuspended in HBSS at a concentration of five times 10 to the seven cells per milliliter, 10 microliters of the cell solution is injected per tibialis, anterior, or TA muscle.
For in vivo imaging, GFP labeled Myoblasts are first transplanted into four to six week old male mice. The mouse is anesthetized with ketamine xylazine, and the procedure is initiated after confirmation of the anesthetic status. To reduce background noise, hair on both hind legs are removed by applying nare to the legs and waiting for 30 seconds before wiping it off.
For the cotton tipped applicator, the legs are then wiped again with a new cotton tipped applicator soaked in distilled water to remove remaining nare and hair to transplant cells into the mouse. First draw five times 10 to the five cells in 10 microliters of HBSS into a Hamilton syringe. With a 30 gauge needle, inject an equal number of cells into three positions along the long axis of the TA muscle, the upper medial and lower part.
After each injection, hold the needle for two minutes before withdrawing it from the muscle in order to prevent leakage along the needle.Tracted. During the waiting period, draw the same amount of cells into another syringe and inject the contralateral TA muscle In the same way after the cell injections, the mouse is immediately placed in the prone position onto a piece of black non fluorescent paper, fastened both feet onto the paper, plantar down and spread the legs 90 degrees apart. The mouse is then placed into the chamber of a night owl, LB 9 81 fluorescence imaging station.
Equipped with a highly sensitive charge coupled camera, one hind leg is positioned in the center of the field of view of the camera. First, take a traditional gray scale photographic picture of the leg. To begin imaging the appropriate emission filter with a specific wavelength for green fluorescent protein is chosen.
Take one image with an exposure of 1, 250 milliseconds followed by another with an exposure of 500 milliseconds. We find that a longer exposure time allows the detection of a weak signal, while a shorter exposure reveals more detail. After one hind leg has been imaged, position the other hind leg in the center of the field of view of the camera image this leg in the same manner as previously shown.
To ensure comparable results, the specimen height should remain unchanged. After imaging, the mouse is transferred back to a cage and its recovery from anesthesia is monitored. For analysis, a region of interest is drawn over the fluorescent signals to measure photons per second.
We also measure the size of the region of interest in number of pixels for an additional quantitative value to acquire longitudinal data, the imaging experiments can be repeated at different days or weeks as needed For the experiments, a fluorescent image of a hind leg of the mouse taken on the day when the GFP labeled cells were injected showed high signal intensity. The signal intensity then dropped on day two and then increased to a stable level by day seven, 14, and 21. When these changes in fluorescence were quantified by measuring signal intensity as photons per second, we observed a marked decrease in signal intensity a day after the injection reflecting a decrease in the number of GFP positive cells.
However, by day seven, 14 and 21, the signal intensity had increased to a stable level indicating that muscle regeneration had occurred. We've just shown you how to use in vivo fluorescence imaging to monitor the progress of regenerating muscle after the injection of GFP labeled cells. Using this method, we're able to study the dynamics of muscle regeneration after the injection of all kinds of fluorescent labeled cells.
Since images can be acquired non-invasively at designated intervals, more accurate longitudinal data can be gathered with fewer animals and lighter workload. When using fixed measurement parameters, images from the same animals over time should be quantitatively comparable. It is extremely important to maintain a clean surface on the skin over the muscle in order to reduce background is absolutely essential to remove all hair covering the legs and to wipe the area thoroughly with cotton swaps or pads.
And remember, don't forget to use the same set of parameters every time to ensure consistency among all your measurements. So that's it. Thanks for watching and good luck with your experiments.
