A method for culturing, identifying and inducing differentiation of Scorpius myoblasts in vitro
A technique for culturing and myoblasts in vitro, applied in the field of cell culture
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Embodiment 1
[0038] The primary culture of the muscle cells of embodiment 1, Xu's flat scorpionfish, concrete method is as follows:
[0039] The tertiary antibody (BI, no.PB180424) is a mixed antibiotic composed of 10000units / mL penicillin, 10mg / mL streptomycin, and 1250units / mL nystatin; 1% of the tertiary antibody means adding 1mL of the tertiary antibody to 99mL of medium. FGFb (Solarbio, no. P00032). Fetal bovine serum (BI, no.04-001-1A). DMEM / F12 (BI, no. 01-172-1ACS). L-15 (Gibco, no. 11415-064). 0.25% trypsin (Gibco, no. 12605-028). Horse serum (BI, no. 04-004-1B).
[0040] 1) Sampling and disinfection of muscle tissue blocks of Scorpius xushii: ①Select juvenile scorpionfish xushii that had just died at the age of 3 to 6 months, and sample the muscle tissue pieces. ②Before sampling, disinfect the surface of the fish with 75% alcohol, wipe the alcohol with toilet paper, and repeat three times to remove bacteria on the surface of the fish as much as possible. ③ When sampling, fi...
Embodiment 2
[0045] The identification method of the subcultured cell species described in Example 1, the present embodiment takes the 30th generation subcultured cells, and the specific method is as follows: 1. The identification of the cell species: by amplifying the 18S rRNA gene of the muscle cell line and the scorpionfish Xu's Compare the homology of 18S rRNA gene in muscle tissue to determine the source of the cell line, design specific primers for 18S rRNA gene, and use the phenol-chloroform method to extract the total DNA of SSCM cells and the total DNA of the muscle tissue of Scorpius xushii. The PCR system is 2.5uL Taq Enzyme buffer (10X), 2uL dNTPs (10mol / L), 0.5uL each primer (10umol / L), 1uL DNA template, 0.2uL taq enzyme, 18.3uL water, the total system is 25uL, after mixing, carry out PCR amplification, amplification The growth conditions were pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 59°C for 30 seconds, extension at 72°C for 1 m...
Embodiment 3
[0059] The method for inducing differentiation of subcultured cells in Example 1: ① 0-24h: set the time for inoculating cells to 0h, and 0-24h is the growth medium, and the growth medium is L-15 medium containing 15% FBS , the cells grow normally at 24h; 24-48h: the growth medium is replaced with a differentiation medium at 24 hours, and the differentiation medium is L-15 medium containing 2% DHS, and cells begin to fuse at 48h; 48-72h : The differentiation medium continued to be cultured, and a large number of cells fused at 72 hours; 72-96h: The differentiation medium was continued to be cultured, and the cell fusion was completed at 96 hours, and almost all of them were multinucleated myotubes; ② For cells cultured in growth medium and differentiation medium Take pictures every 24 hours to record the process from mononuclear muscle cells to fusion into multinucleated muscle fibers; ③ use Hoechst and phalloidin to stain the nucleus and cytoplasm of the cells at 96 hours respe...
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