The invention provides a mouse 
skeletal muscle satellite cell separation and culture method. The mouse 
skeletal muscle satellite cell separation and culture method comprises following steps: 1, mice are killed through 
intraperitoneal injection of pentobarbital 
sodium, and are immersed in 75% 
alcohol for disinfection; 2, the mice are fixed on plates with the venter posterior surfaces upward, hind limb 
skin is peeled under aseptic conditions, and thighbone 
muscle tissues are collected; 3, in an aseptic operation bench, PBS washing is carried out for three times, surface blood vessels, tendons, and connective tissues are removed; 4, the processed tissues are 
cut into pieces, and are subjected to 
digestion with preheated 
trypsin and IV type 
collagenase respectively; 5, 
digestion is stopped, 
centrifugation is carried out, and an obtained supernatant is removed; 6, a 
cell filtrate is collected, and trypan-blue 
drying detection is adopted to detect 
cell activity; 7, after 
cell counting, a complete medium is adopted for resuspension 
inoculation into a coated culture flask for culture at 37 DEG C at 5% CO2 environment; 8, differential attachment method is adopted to remove fibrocytes; 9, 
cell morphology is carried out; 10, 
ELISA method detection is carried out; and 11, RT-PCR detection is carried out. The mouse 
skeletal muscle satellite cell separation and culture method is simple in operation, and is high in 
survival rate; the number of obtained cells is large; the mouse skeletal 
muscle satellite 
cell separation and culture method is an ideal method, and is capable of providing experiments with reliable cell resources.