After euthanizing the mouse, dissect TA and GA muscles from both hind limbs and place them in the lid of a Petri dish. Use scissors to cut the tissue into a minced slurry. Transfer the minced tissue to a 50-milliliter tube containing five milliliters of ice cold dissociation buffer, and keep it on ice.
Once the sample tubes are heated at 37 degrees Celsius, incubate them for 45 minutes on rotation in an incubator. Add 10 milliliters of wash media to the sample tubes and vortex them. Centrifuge the tubes, and aspirate the contents to four milliliters.
Next, add 0.5 milliliters each of collagenase and dispase to the cells. Then, vortex and incubate them. After incubation, centrifuge the digested sample and resuspend the pellet with a five-milliliter pipette.
Next, pre-wet the 40-micrometer cell strainers placed on 50-milliliter tubes with five milliliters of wash media. Using a five-milliliter syringe with a 20-gauge needle, aspirate and eject the cell suspension 10 times. Then, strain the cell suspension through the pre-wetted 40-micrometer cell strainer.
After collecting and straining the remaining cells with 10 milliliters of wash media, pellet the cell suspension by centrifugation, aspirate the supernatant from the tube, and gently flick the pellet to loosen it.
