Normal rat primary aortic smooth muscle cell culture

PriCells - normal rat primary aortic smooth muscle cell culture
First, experimental reagents
1. Medium: PriCells Medium + 10% FBS + 1% P/S + PriCells Supplement
2. Cryopreservation solution: PriCells 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: PriCells Isolation of Primary Cell Kit
6, detection reagent: actin, a-SMA or Desmin antibody, fluorescently labeled secondary antibody, 4% paraformaldehyde
Second, the experimental equipment
1. Petri dish
2, culture bottle
3, direct cut and eye scissors
4, ophthalmology and hemostats
5, glass dropper
6, beaker
7, 15ml centrifuge tube
Third, the experimental process
Adult rats, anesthetized, aseptically obtained aortic vessels
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Wash the blood vessels in 1 × PBS (pH 7.4) and peel off the tissue attached to the extravascular
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The blood vessels were cut longitudinally, the intima was upward, and the endothelium was scraped laterally with blunt forceps, and the endothelial cells were removed and washed repeatedly with 1 × PBS (pH 7.4).
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The blood vessels were scraped laterally with forceps, the middle layer was scraped off, the middle membrane was collected and washed with PBS, a small amount of medium (PriCells) was added, and the blood vessels were cut into small pieces of 1 × 1 mm 3 and washed with 1 × PBS (pH 7.4). Times
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Digestion solution (PriCells) was added to the tissue block, transferred to a 15 ml centrifuge tube and digested at 37 ° C in a water bath for 15 min, shaken at intervals of 2-3 min, allowed to stand for 1 min, and the digestive juice was collected and repeatedly digested 3-4 times.
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Collect the digestive juice and add the digestion stop solution (PriCells) to stop the reaction.
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Centrifugation, 1000rmp, 10min, discard the supernatant
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Resuspend, count cells
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Inoculation density is 5 × 10 5 /ml
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Culture at 37 ° C, 5% CO 2
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. Materials: Normal adult rats were intraperitoneally injected with 5% phenobarbital anesthesia, 75% alcohol soaked, and the rat aorta was quickly removed under sterile conditions.
3. Material pretreatment: The obtained blood vessel was repeatedly washed in 1 × PBS (pH 7.4) containing 1% P/S to remove blood stains outside the blood vessel, the washing liquid was clarified, and the outer membrane surface was peeled off with a sterile forceps. Residual tissue. Wash with PBS.
4. Removal of endothelial cells: the blood vessels were longitudinally cut, the intima was faced upward, the sterile tweezers repeatedly scraped the intima layer 4-5 times along the central axis, the endothelial cells were removed, and washed 3 times with 1 × PBS (pH 7.4). .
5, separation of the middle membrane: will remove the blood vessels of the intima, continue to scrape the separation of the membrane, remove the outer membrane, use the ophthalmic scissors to cut the inner membrane into small pieces of 1 × 1mm3, add 1 × PBS (pH 7.4), transfer The cells were washed 3 times with PBS in a 15 ml centrifuge tube, and the precipitate was collected by centrifugation.
6. Digestion: Digestive enzyme solution was added to the washed endometrial fragments, and the centrifuge tube was placed in a 37 ° C water bath for 15 min.
7. Stop digestion: aspirate the digestive juice into a new centrifuge tube, add the digestion stop solution according to 1:1, and stop the enzyme reaction; the remaining tissue continues to be added to the digestive juice, digested for 15 min, and repeatedly digested for 3 times.
8. Collect the resuspended cells: collect the digested solution after the suspension in a centrifuge tube, centrifuge at 1000 rpm for 10 min, discard the supernatant, resuspend the culture medium, and stain the cells.
9. Culture cell density: The cell density was adjusted and inoculated into the culture flask at 5 × 105 cells/ml.
10. Culture: Place in a 37 ° C, 5% CO2 incubator.
V. Cell identification
1. Microscopic identification: Under the phase contrast microscope, the cells are long fusiform and have good light transmission. Typical peak-wave-like growth between cells is characterized by contact inhibition.
2. Immunohistochemical identification: Smooth muscle cells were identified by actin, a-SMA or Desmin immunofluorescence staining.
1) Cell climber. 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.
2) Cell fixation: The cells were washed with 1 × PBS (pH 7.4), then placed in 4% paraformaldehyde, fixed for 10 min, and naturally dried in the air.
3) Specific antibodies: Dilute the antibody as required by dilution according to the instructions, add the antibody, and incubate at 37 ° C for 60 min.
4) Washing: Wash 1 × PBS (pH 7.4) 3 times × 15 minutes, and dry.
5) Labeled antibody: Add fluorescently labeled antibody and incubate for 30 min at 37 °C.
6) Washing: Wash 1 × PBS (pH 7.4) 3 times × 15 minutes, and dry.
7) Cover: Seal the tablet.
8) Microscopic examination: Under the fluorescence microscope, the cytoplasm of smooth muscle cells showed strong brown-red fluorescence.
Six, matters needing attention
1. In order to maintain cell viability, the rats are suitable for taking materials after anesthesia. Rats should be sterilized strictly, sterilized aseptically, and the thoracic cavity should be opened to exchange another device for aortic vessels.
2, pay attention to wash the residual blood in the blood vessels as much as possible, to avoid the impact of blood cells and certain serum components on cell adhesion.
3. The blood vessels are composed of a three-layer membrane structure of the intimal layer, the middle layer and the outer membrane layer, and the vascular smooth muscle cells are mainly distributed in the middle layer of the blood vessel, and the inner membrane is obtained by mechanical scraping to remove the inner membrane to separate the cells. Cells scraped off the endocardial surface can be collected and counted to culture aortic endothelial cells.
4, enzymatic digestion method for the isolation of vascular smooth muscle cells, enzyme digestion is easy to damage cells, affecting the adherence of smooth muscle cells, so the digestion process should be based on the composition of the isolated cell source tissue, select the appropriate enzyme, and strictly control the enzyme Digestion concentration and digestion time.
5. Strictly control cell culture conditions. Including the quality and concentration of cell culture fluids (culture medium, growth factors, serum, antibiotics).
6. The inoculation amount of the subculture cells was 5 × 10 4 (25 cm 2 flask), and the cell doubling time was 48 hours. Cell subculture is best for 5-8 generations, the number of passages is increased, the cell volume is increased, the cells are easy to fuse with each other, the boundary is unclear, the cells are firmly attached, and the passage time is prolonged.
Seven, cell picture

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