Method for inducing skeletal muscle satellite cells to generate lipid droplets
A satellite cell and skeletal muscle technology, applied in the field of cells, can solve problems such as poor pork quality, and achieve the effects of improving pork quality, convenient operation and simple method.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0038] The specific steps for obtaining pig skeletal muscle satellite cells are as follows:
[0039] Slaughter the newborn piglets, quickly take the half-bond muscles and back muscles of each individual, and immediately put them in Hanks solution, pack them and do the next step;
[0040] Cut out the required skeletal muscle in the clean bench, wash with PBS containing 1% double antibody, and remove connective tissues such as fat, muscle bonds, and sarcolemma as much as possible;
[0041] Cut into pieces to about 2 mm3 and transfer to a centrifuge tube, blow and beat with PBS 3 times, settle naturally for 5 minutes, and discard the supernatant; add 0.2% collagenase, digest in a 37°C water bath for 50 minutes, shake once every 5 minutes, and wash with PBS three times after the end. Centrifuge at 1000r / min for 10min, discard the supernatant; then digest with an appropriate amount of 0.25% trypsin in a 37% water bath for 20min, shake once every 5min, and immediately add an equal v...
Embodiment 2
[0044] After the pure skeletal muscle satellite cells obtained in Example 1 were fixed in 4wt% paraformaldehyde cell fixative for 15 minutes, the cells were permeabilized with 0.25% TritonX-100, followed by rinsing with PBS for 3 times, and then adding 3wt% BSA Block for 1 hour, add primary antibody PAX-7, incubate overnight at 4°C, absorb primary antibody PAX-7, wash with PBST three times, add fluorescent secondary antibody, incubate for one hour in the dark, then observe under an inverted fluorescence microscope, as shown figure 2 shown.
[0045] Depend on figure 2 It can be seen that PAX-7 is positively expressed in green fluorescence, and the nuclei are in blue. This proves that the cells obtained in Example 1 are indeed skeletal muscle satellite cells.
Embodiment 3
[0047] To induce adipogenesis of porcine skeletal muscle satellite cells, the specific steps are as follows:
[0048] A. Collect skeletal muscle satellite cells from the dorsal muscle of newborn piglets 1-5 days old, and purify the cells through differential attachment. After the purified skeletal muscle satellite cells are adhered to 60%-70% of the confluence, adipogenic induction is carried out ;
[0049] B. On day 1-2 of induction of differentiation, add 1 μmol / L dexamethasone, 5 μg / mL insulin, 0.5 mmol / L 3-isobutyl-1-methylxanthine and 1% to DMEM / F12 basal medium Double antibody, cultivated for 2 days;
[0050] C. On the 3rd-4th day of induction of differentiation, add 5 μg / mL insulin, 10uM rosiglitazone and 1% double antibody to the DMEM / F12 basal medium, and culture for 2 days;
[0051] D. On day 5-6 of induction of differentiation, add 1 μmol / L dexamethasone, 5 μg / mL insulin, 0.5 mmol / L 3-isobutyl-1-methylxanthine and 1% to DMEM / F12 basal medium Double antibody, cult...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More