Hi,
I am doing experiment in stem cells so I need to isolate the satellite cell from mice skeletal muscle. So i need the suggestions how could successfully isolate the pure satellite cells from mice skeletal muscle and what are the critical steps and precautions.
Note: I am using preplate technique but could not desired aim yet.
Thanks in advance
Arif
Have you seen the "Isolation and Culture of Satellite Cells from Mouse Skeletal Muscle" chapter by Antonio Musarò and Silvia Carosio in Paolo Di Nardo et al. (eds.), Adult Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1553, DOI 10.1007/978-1-4939-6756-8_12, See https://link.springer.com/content/pdf/10.1007%2F978-1-4939-6756-8_12.pdf.
Second that answer otherwise this is a review of the current techniques.
Isolation and Culture of Muscle Stem Cells.
Mozzetta C. Methods Mol Biol. 2016.
Hi there,
this is the standardized protocol we use in our lab. using MACS (Magnetic Activated Cell Sorting):
hind limb muscles were gently isolated from mice, non-muscle tissue was removed and muscles were minced and subjected to enzymatic dissociation for 45 min at 37°C. The enzymatic mix was composed by 2μg/ml collagenase A (Roche cat#10103586001), 2,4 U/ml dispase II (Roche cat#04942078001) and 0,01mg/ml DNase I (Roche cat#04716728001) diluted in D-PBS with Calcium and Magnesium. Enzymatic dissociation was stopped by addition of Hank’s Balanced Salt Solution (HBSS) and the cell suspension was filtered through a 100, 70, 40 and 30-μm cell strainer. Cells were incubated with the appropriate antibodies conjugated with magnetic micro beads and isolated using the MACS separation technology. The antibodies used for lineage negative were CD45 (Miltenyi cat#130-052-301) and CD31 (Miltenyi cat#130-097-418). The CD45-/CD31- cells were further stained with a7-integrin micro-beads antibody (Miltenyi cat#130-104-261). SCs were selected as CD45-/CD31-/a7-integrin+ cells and cell purity was confirmed by Pax7 expression.
Good Luck