Preparation method of tissue single-cell suspension
A single-cell suspension and tissue technology, which is applied in the field of genetic testing, can solve the problems that restrict the establishment of cell culture cell lines and functional gene verification research, and the inability to obtain single-cell suspensions of cell viability and tissue, so as to achieve low-cost and high-efficiency acquisition Effect
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[0027] A method for preparing a tissue single-cell suspension, comprising the following steps:
[0028] 1) preparing a reagent and a culture medium, the culture medium comprising two kinds of culture medium which are respectively a serum-free medium and a complete medium, preparing a cell cleaning solution, and preparing a collagenase II dissociation stock solution;
[0029] 2) Tissue pretreatment, add an appropriate amount of 1*DPBS solution in a sterile RNase-free cell culture dish, and cut the isolated tissue into 0.5-1mm 2 After the small pieces are placed in a sterile RNase-free cell culture dish containing 1*DPBS solution, rinse 1-2 times with 1*DPBS solution until the blood, fat layer and other excess tissues are removed;
[0030] 3) Preliminary digestion and dissociation of the tissue, collagenase II dissociation stock solution, added to a clean 15ml tube, and marked on the tube wall, using tweezers to clamp the small pieces of tissue treated in step 2) into the collag...
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[0047] Step 1: Configure reagents and media
[0048] 1. Prepare the following two media respectively
[0049] (1) Serum-free medium: subpackaged RPM1640 or DMEM / F12 medium contains 1% Penicillin-Streptomycin; add 1ml Penicillin-Streptomycin to 99ml RPM1640 or DMEM / F12 medium
[0050] (2) Complete medium: subpackaged RPM1640 or DMEM / F12 medium contains 10% FBS and 1% Penicillin-Streptomycin; add 10ml FBS and 1ml Penicillin-Streptomycin to 89ml RPM1640 or DMEM / F12 medium
[0051] 2. Prepare cell washing solution: 1*DPBS solution containing 0.04% BSA: add 0.04gBSA to 100ml 1*DPBS solution
[0052] 3. Prepare collagenase II dissociation stock solution: dissolve 20 mg of collagenase II freeze-dried powder (Sigma, product number C6885-1G) in 10 ml of serum-free RPM1640 or DMEM / F12 medium, and divide after completely dissolving. Put it into a 1.5ml centrifuge tube and store it in a -20°C freezer to avoid repeated freezing and thawing.
[0053] Step Two: Tissue Pretreatment
[005...
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