To begin, spray the sacrificed 10-week-old C57 black 6J male mouse thoroughly with 70%ethanol. Using forceps, remove the skin from the hindlimb and dissect all limb muscles surrounding the femur, tibia, and fibula. Place the harvested limb muscles into a two milliliter tube containing one milliliter of muscle isolation buffer.
And using scissors, mince the harvested muscles. Transfer the minced muscle suspension into a 50 milliliter tube containing 18 milliliters of muscle isolation buffer. Close the tube tightly, seal it with laboratory film, and place it horizontally in a shaking water bath.
After 30 minutes, add five milligrams of type I collagenase and maintain the tube for another 30 minutes. After pipetting the muscle suspension up and down 10 times, centrifuge and discard the supernatant, leaving five milliliters of the medium in the tube. Pipette the suspension onto the successive cell strainers and collect the flow-through into the 50 milliliter tube.
Centrifuge the suspension and discard the supernatant until only 100 to 200 microliters remain in the tube. Resuspend the pellet in two milliliters of red blood cell lysis buffer and incubate on ice for three minutes. Centrifuge again and remove the supernatant from the tube using a 20 to 200 microliter pipette.
Resuspend the pellet in 100 microliters of cold FACS buffer and place the tube on ice. After removing 10 microliters of cell suspension for the negative control, centrifuge the remaining 90 microliters and discard the supernatant before incubating the cells with 400 microliters of fixable viability stain diluted in serum-free DMEM for 15 minutes at room temperature. After centrifugation, add 100 microliters of FACS buffer and gently invert the tube thrice.
Centrifuge again and add 100 microliters of primary antibody mixture into the tube. After 30 minutes of incubation in the dark, centrifuge and discard the supernatant. Then add 500 microliters of PBS to wash the cells.
Centrifuge again and remove the supernatant before resuspending the pellet in 500 microliters of FACS buffer. Transfer the suspension to a five milliliter tube. Briefly vortex the cell suspension before processing it on a flow cytometer.
Collect the selected cells in the five milliliter coated tube containing FACS buffer. 75%of the mononucleated cells identified based on their size and granularity were negative for the fixable viability stain marker leading to an average of 34.3%of live cells. About 3%of single living cells were negative for monocyte, endothelial, leukocyte, and erythroid specific markers.
These negative cells were then selected according to their expression of CD34 and ITGA7 markers. A final gating for CXCR4 was performed to select putative satellite cells. And from the CD34-positive ITGA7-positive cells, 80%were found to be positive for CXCR4.
