Separation and culture method of ovarian granular cells of northern Guizhou Ma goats
A Qianbei hemp sheep and granular cell technology, applied in the field of cell biology, can solve the problems of few cleaning times, no clear regulation of cleaning times, probability of cell contamination, etc., to improve accuracy, easy operation, and fewer dead cells. Effect
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Embodiment 1
[0040] The method for isolating and culturing ovary granulosa cells of Qianbei Ma sheep comprises the following steps:
[0041] (1) The adult purebred Qianbei Ma sheep ewes were sacrificed by neck bleeding, the ovaries and follicles were separated, and 75% alcohol was used to sterilize for 35 minutes at 110°C and 100kPa, and 1:1 bluegrass was added. and streptomycin mixed solution in PBS buffer to remove blood stains, rinsed 3 times and then transferred to a beaker filled with PBS buffer pre-cooled to 4°C, and quickly transferred to a sterile room within 4 hours. During the transfer process, the temperature was kept at 4°C;
[0042] (2) Take out the ovaries and follicles treated in step (1), soak them in 75% alcohol for 30 seconds, and press 75% alcohol-physiological saline-sterilized PBS buffer solution (containing 1:1 penicillin and streptomycin solution) - Sequential washing of DME / F12 medium 3 times;
[0043] (3) Put the ovaries and follicles treated in step (2) in a pet...
Embodiment 2
[0048] The method for isolating and culturing ovary granulosa cells of Qianbei Ma sheep comprises the following steps:
[0049] (1) The adult purebred Qianbei Ma sheep ewes were sacrificed by neck bleeding, the ovaries and follicles were separated, sterilized with 75% alcohol and sterilized at 120°C and 100kPa for 20 minutes, and added 1:1 green and streptomycin mixed solution in PBS buffer to remove blood stains, washed 3 times, then transferred to a beaker filled with PBS buffer pre-cooled to 4°C, and quickly transferred to a sterile room within 5 hours. During the transfer process, the temperature was kept at 8°C;
[0050] (2) Take out the ovaries and follicles treated in step (1), soak them in 75% alcohol for 30 seconds, and press 75% alcohol-physiological saline-sterilized PBS buffer solution (containing 1:1 penicillin and streptomycin solution) - Sequential washing of DME / F12 medium 5 times;
[0051] (3) Put the ovaries and follicles treated in step (2) in a petri dish...
Embodiment 3
[0056] The method for isolating and culturing ovary granulosa cells of Qianbei Ma sheep comprises the following steps:
[0057] (1) The adult purebred Qianbei Ma sheep ewes were sacrificed by neck bleeding, the ovaries and follicles were separated, sterilized with 75% alcohol and sterilized at 115°C and 90kPa for 30 minutes, and added 1:1 green and streptomycin mixed solution in PBS buffer to remove blood stains, washed 3 times, then transferred to a beaker filled with PBS buffer pre-cooled to 4°C, and quickly transferred to a sterile room within 4.8 hours. Keep at 7°C;
[0058] (2) Take out the ovaries and follicles treated in step (1), soak them in 75% alcohol for 30 seconds, and press 75% alcohol-physiological saline-sterilized PBS buffer solution (containing 1:1 penicillin and streptomycin solution) - Sequential washing of DME / F12 medium 5 times;
[0059] (3) Put the ovaries and follicles treated in step (2) in a petri dish filled with 100ml of 10% complete medium, where...
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