A method for isolating spermatogonial stem cells from turbot
A technology of spermatogonial stem cells and separation method, which is applied in the field of turbot spermatogonial stem cell separation, can solve the problems of serious inbreeding, decline, frequent occurrence of diseases, etc., and achieve the effect of high purity, high density, and complete morphological structure
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[0019] (1) Collect 2-3 tails of turbot weighing 300-500g at the age of about 1, separate the gonads of the male fish substage in the live body, and put them into 100ml of pre-cooled 0.1M PBS;
[0020] (2) Cut the gonads into about 0.5g portions on crushed ice, and cut them into pulp;
[0021] (3) Add 1 mL of L-15 culture medium containing 1% double-antibody (penicillin / streptomycin) to wash, centrifuge at 200 g for 5 min at 4°C, discard the supernatant, and repeat twice;
[0022] (4) Put into L-15 culture solution containing 1mL 0.2% trypsin, 5% fetal bovine serum, and 0.05% NaseI and incubate at 25°C for 2 hours, and gently blow and beat with a pipette to disperse the gonad cells;
[0023] (5) filter with a nylon mesh with a pore size of 42 μm, remove undissociated cell masses, and collect the filtrate;
[0024] (6) Add 3mL-15 culture solution to wash, blow and mix with a pipette, centrifuge at 200g at 4°C for 5min, discard the supernatant, repeat twice;
[0025] (7) Add 1m...
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