This protocol describes a method for the simultaneous isolation of fibro adipogenic, progenitors, and muscle stem cells using fluorescence activators of sorting. Pure populations of these cells are a prerequisite for understanding their biological role in physiological and pathological conditions. Using this technique, Forbes and muscle stem cells were identified and isolated from either armpit or injured muscle.
The high filled impunity of Forbes and muscle stem cells have allowed us to use this cells for different downstream techniques such as saccharine transplantation and mudial mix analysis. To begin, place the euthanized mouse supine on a dissection pad and spray it with 70%ethanol To avoid contamination use forceps to pinch the center of the mouse belly skin and cut approximately one centimeter opening horizontally. Grasp the wound edges and disk in the mouse by pulling the opening in the opposite directions to uncover the muscles underneath, expose one side of the hind limb at the time.
Before collecting muscles, Remove intermuscular fat between the hamstrings in the proximal end of the quadriceps to improve the isolation of fibro adipogenic progenitor cells and muscle stem cells. Next, without damaging the tissue use sharp forceps to break and peel off the fascia to expose the underneath tibialis anterior or TA and extensor digitorum longus or EDL muscles. Run the sharp tip of the forceps underneath the TA EDL muscles to detach the muscles from the tibia.
Cut off the tendons, attaching the TA EDL to the ankle and while holding it with forceps, trim the muscles with scissors along the longitudinal line of the TA EDL. Cut the tendons around the knee to detach the whole TA EDL muscles. Place the muscles in a six centimeter dish kept on ice.
To isolate gastrocnemius muscles cut all tendons at the ankle and detach the muscles from the tibia and fibula. Cut around the knee and transfer the muscles to the dish. Peel off the fascia around the quadriceps and separate it from the femur by running the sharp tip of the forceps between the muscle and bone.
Cut the tendons around the knee and trim the rest of the muscle by cutting along the femur while holding the quadriceps with forceps at the distal end. Place the muscles in the six centimeter dish. Detach the hamstrings and remaining muscles around the femur and transfer those to the dish.
Collect the hind limb muscles in a dish kept on ice. Aspirate wash medium from the dish and add one to two milliliters of muscle dissociation buffer to keep the muscles moist. Next, mince muscles thoroughly Use a scalpel to tear and slice the muscle and then use scissors to keep cutting the muscle into small pieces for one to two minutes until obtaining a slurry paste of well minced tissue.
Then transfer the minced muscles to the conical tube containing muscle dissociation Buffer. Seal the tubes with laboratory film and incubate in a 37 degree Celsius water bath with agitation for one hour. After one hour, remove the tube from the shaker and fill it to 50 milliliters With wash medium, gently invert the tube twice to ensure mixing.
Centrifuge the cells in a swinging bucket Rotor at 250 times G for five minutes at four degree Celsius. Transfer 42 milliliters of supernatant into two new tubes and leave eight milliliters in the original tube. Fill the new tubes containing 21 milliliters of the supernatant up to 50 milliliters with wash medium.
Then centrifuge the cells at 350 times G for eight minutes at four degrees Celsius. Aspirate the supernatant and keep the pellets on ice. Next, add one milliliter of collagenases two stock and one milliliter of dis based stock in the original tube containing eight milliliters of muscle dissociation buffer.
Using a five milliliter serological pipette resuspend the pellet 10 times without clogging. Eject the solution toward the wall of the tube, avoiding the formation bubbles. Seal the tubes with laboratory film and incubate in a 37 degrees Celsius water bath with agitation for 30 minutes.
After 30 minutes, remove the tube from the shaker aspirate and eject the muscle suspension 10 times through a 10 milliliter syringe with a 20 gauge needle, fill each tube up to 50 milliliters with wash medium and invert the tube. Then centrifuge at 250 times G for five minutes at four degree Celsius and transfer the supernatant into a new tube. Leaving eight milliliters in the original tube.
Fill the new tube up to 50 milliliters with wash medium, again centrifuge the cells at 350 times G for eight minutes at four degree Celsius. Remove the supernatant and keep the pellet on ice. Place a 40 micron nylon cell strainer in the original 50 milliliter conical tube containing eight milliliters of wash medium and pre-wet the strainer with one to two milliliters of wash medium.
While holding the strainer, Use a 10 milliliter pipette to resuspend the pellet gently. Filter the suspension through the cell strainer back into the same tube to minimize cell loss. Filter the other tube pellets back into the original tube by adding four to five milliliters of wash medium to each tube to resuspend the pellets and transfer the solution to the self strainer positioned in the original tube.
After collecting all the pellets into the original 50 milliliter tube rinse the self strainer with another four to five milliliters of wash medium. Use a 1000 microliter pipette to collect all the liquid from the underside of the self strainer. Fill each tube with wash medium up to 50 milliliters and invert the tube.
Centrifuge the tube at 250 times G for five minutes at four degrees Celsius. Remove the supernatant immediately without disturbing the pellet and resuspend the pellet in 600 microliters of wash medium. Transfer the suspension to a two milliliter micro centrifuge tube.
Add antibodies CD 31 APC CD 45 APC SCA one Pacific Blue VCAM one biotin and alpha seven Integrin PE into a two millimeter tube containing cell suspension. Gently mix and incubate the cells in a rotating shaker at four degree Celsius for 45 minutes protecting them from light. After incubation, fill all the tubes up to two milliliters with wash medium and centrifuge at 250 times G for five minutes at four degree Celsius.
Remove the supernatant and resuspend the pellet in 300 microliters of wash medium. Next, add streptavidin antibody into tubes and incubate the cells in a rotating shaker at four degree Celsius for 20 minutes. After incubation, fill tubes containing streptavidin antibody to two milliliters with wash medium.
Then, centrifuge the tubes and aspirate the supernatant completely. After the second wash, resuspend the cells in the experimental tube in 500 microliters of wash medium. Pre-wet a cell strainer cap of a five milliliter polystyrene round bottom tube with 200 microliters of wash medium.
Transfer the cell suspension from the experimental tube into the five milliliter polystyrene round bottom tube and allow it to filter by gravity. Print the two milliliter tube containing the experimental sample suspension with 300 microliters of wash medium and pass it through the same strainer cap. Use a 200 microliter pipette to collect all the liquid from the underside of the cell strainer.
Seal with a cap, leave tubes on ice and protect them from light. The gating strategy adopted for the isolation of fibro adipogenic progenitor and muscle stem cells is shown here. The PD GFR alpha E G F P knock in reporter mice expresses the H two B E G F P fusion gene from the endogenous PD G FFR alpha locus, allowing efficient and specific labeling of the PD G GFR alpha lineage.
The purity of the isolated cell populations was confirmed by immuno staining of co-culture GFP positive fibro adipogenic progenitor and muscle stem cells with PAC-seven antibodies. GFP positive fibro adipogenic progenitor did not express the PAC-seven marker, while muscle stem cells reacted with this antibody. All GFP expressing cells were positive for SCA one and negative for CD 31, CD 45 VCAM one and alpha seven integrin.
The fibro adipogenic progenitor and muscle stem cells isolated from mice injured with intramuscular injections of no toxin seven days post-injury are shown. As muscle stem cells are activated an increase in cellular size. It is important to adjust the F SCA and SS CA parameters and expand the gate to include those cells in the center.
Quantification of fibro adipogenic progenitor and muscle stem cells in uninjured and seven days post-injury TA muscles are shown here. Although the peak and proliferation was observed between days two and three a low rate of proliferating cells was still appreciable seven days after injury. Performing proper muscle mincing is of critical importance to release a single cells.
Furthermore, mononucleotide cell isolation is achieved by running the suspension to a 10 milliliter syringe with a 20 gauged needle. This technique will help scientists to study the biological transcriptional and epigenomic profile of Forbes and muscle stem cells in normal condition and during the deeper stages of muscle regeneration.
