First, experimental reagents
1. Medium: Primary Cell Medium + 10% FBS + 1% P/S + Primary Cell Supplement
2. Cryopreservation solution: Primary Cell Medium + 20% FBS + 10% DMSO
3. Washing solution: 1 × PBS (pH 7.4 ) + 1% P/S
4. Staining solution: 0.4% Trypan Blue
5. Digestive juice: Isolation of Primary Cell Kit
6. Detection reagent: anti-mouse Fibronectin antibody, fluorescently labeled secondary antibody, ethanol-acetone mixture (1:1)
Second, the experimental equipment
1. Petri dish
2, culture bottle
3, direct cut and eye scissors
4, ophthalmology
5, glass dropper
6, 15ml centrifuge tube
7, 200 mesh screen
Third, the experimental process
Take the skin piece and cut it into a piece of leather with a thickness of 0.5mm↓
The skin is immersed in 75% alcohol for disinfection↓
1 × PBS (pH 7.4 ) to clean the skin twice
Cut the skin into (3-5) mm × (10-15) mm size ↓
Digested in digestive juice at 4 ° C overnight
Peel off the epidermis and scrape off other tissues under the dermis
Cut the tissue block into 1mm3 or so
Add the digestive juice and digest at 37 ° C until the digestive juice is cloudy.
Stop digestion and suspend through 200 mesh screen
Collect the cell suspension and centrifuge at 800 rpm for 5 min.
↓
Resuspend the cells and repeat the centrifugation
Resuspend the cells, try to stain with Trypan Blue, and inoculate the flask in a number of 5×105.
37 ° C, 5% CO 2 culture
Fourth, experimental operation
1. The culture bottle is pre-coated. The flask was pre-packaged one day before the test, placed in an ultra-static table or dried in an oven at 45 ° C (remember to keep it sterile), placed in a refrigerator at 4 ° C, and set aside.
2, taking materials: take the skin from the mouse, cut into a skin with a thickness of about 0.5mm;
3. Material pretreatment: immerse the skin in 75% alcohol for disinfection; wash twice with 1 × PBS (pH 7.4) (about 5 min each time) to remove blood cells;
4, preliminary digestion: cut the skin into (3) mm × (10) mm size, remove the subcutaneous tissue; cut the cut skin in the digestive juice at 4 ° C digestion overnight; the next day, remove the digested cells Use tweezers to peel off the epidermis as much as possible and scrape off other tissues under the dermis;
5, digest again: after treatment, cut the tissue block into about 1mm3, add digestive juice, shake at 37 °C to digest the turbid liquid (about 1h);
6, stop digestion: stop the enzyme reaction, the suspension through the 200 mesh screen;
7. Collect the resuspended cells: collect the cell suspension, centrifuge at 800 rpm for 5 min; discard the supernatant, resuspend the cells in the medium, and repeat the centrifugation;
8. Cell density count: The medium was resuspended in cells, counted by 0.4% Trypan Blue staining, and seeded in a culture flask at a number of 5 x 105.
9. Culture: Place in a 37 ° C, 5% CO2 incubator.
V. Cell identification
1. Microscopic identification: Under the phase contrast microscope, it can be seen that the cells are fusiform or flat star-shaped with protrusions. The cells have good light transmission.
2. Immunohistochemical identification: detection using Fibronectin-related antigen.
3. Cell climbing. The washed coverslips were placed in a 6-well culture plate, and the cells were seeded at 3×10 4 cells/well. At 48 hours, the cells were overgrown, and the coverslips covered with cells were taken out with tweezers for use.
4. Cell fixation: The cells were washed with PBS, then placed in a mixture of ethanol and acetone, fixed for 10 min, and naturally dried in the air.
5. Specific antibody: Dilute the antibody according to the dilution requirements, add the antibody, and incubate at 37 ° C for 60 min.
6. Wash: Wash 1 × PBS (pH 7.4) 3 times × 15 minutes, and dry.
7. Labeled antibody: Add fluorescently labeled antibody and incubate at 37 ° C for 30 min.
Washing: Wash 1 × PBS (pH 7.4) 3 times × 15 minutes, and dry.
8. Cover: Seal the tablet.
9, microscopic examination: observed under a fluorescent microscope.
Six, matters needing attention
1. Remove the coat on the skin tissue when selecting the material, or use the suckling mouse that has not grown.
2, pay attention to cut the skin into (3) mm × (10) mm size, if too large is not conducive to digestion, too small is not conducive to the physical separation of the dermis and epidermis.
3, excessive digestion damage seriously affects the growth of fibroblasts, so the enzyme digestion concentration and digestion time should be strictly controlled.
4. Strictly control the culture conditions of fibroblasts. Including the quality and concentration of cell culture fluids (culture medium, growth factors, serum, antibiotics).
5. Regarding the culture of the bottle or not, there is literature research showing that there is no particularly large difference between the coated and uncoated. In the experiment, it is possible to consider whether or not to coat.
6. The inoculation amount of the subculture cells was 5 × 104 (25 cm 2 culture flask), and the cell doubling time was 48 hours. The cell subculture was best for 8 generations, the number of passages increased, the cell volume increased, the intercellular space increased, the cells adhered firmly, and the passage time was prolonged.
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