Satellite Cells: Regenerative Mechanisms and Applicability in Muscular Dystrophy

Table 2

Summarized protocol for isolation and culture of SCs on Matrigel.

Step	Procedure
Obtaining the whole cell suspension
Muscle collection	Properly dissect an entire muscle from the euthanized animals and transfer to ice-cold PBS for washing
Muscle preparation	(i) Dissect the muscle under light microscope in order to remove connective tissue, blood vessels, nerve fibers, and adipose tissue; (ii) Cut and mince tissue for enzymatic digestion; (iii) Digest with 0.2% collagenase with 10% fetal bovine serum (FBS) in DMEM medium
Obtaining a single-cell Solution	(i) Triturate mixture in order to obtain a single cell solution in up to 50 mL of DMEM with 2% FBS; (ii) Pass the cell suspension through a 70 μm cell strainer
Cell counting and concentration	(i) Count cells on hemocytometer; (ii) Perform successive centrifugations to attain cells in a 200 μL suspension; (iii) The usual number of cells obtained from one mouse is of 2 × 10^6; (iv) Suspension is to be done in 2% FBS in DMEM
Isolation
SCs antibodies	(i) 1 μL of antibodies against CD31-PE, CD45-PE, integrin-7, and Sca-1-PE should be added to the 200 μL cell suspension; (ii) Incubation for 30 minutes on ice; (iii) Wash cells with 1 mL of 2% FBS in DMEM by centrifuging at 2000 rpm for 3 minutes at 4°C; (iv) Resuspend cells in 200 μL of the 2% FBS and DMEM solution
Magnetic beads addition	(i) Add 10 μL of anti-PE magnetic beads; (ii) Incubate on ice for 30 minutes
Cell washing and suspension on MACS buffer	(i) Wash the cells twice in 1 mL of MACS buffer; (ii) Resuspend in 1 mL of MACS buffer
Cell suspension elution and collection	Proceed MACS protocol by eluting the cell suspension through the column in order to obtain only the cells marked by the SCs-related antibodies
Culture
Resuspension on myoblast medium	Resuspend cells in medium containing; (i) Hams F10 medium; (ii) 20% FBS; (iii) Basic fibroblast growth factor (bFGF)
Plating	Cells should be plated in Matrigel-coated 10 cm plates containing 8 mL of the appropriate medium
Maintenance	Medium should be changed every 2 days and passaging is to be done before reaching 50% confluence