Rat scleral epithelial cells

Experimental report:

1、 Separation and cultivation:

1. Under sterile conditions, extract atrial tissue from 1-3 day old SD rats, wash the tissue block twice with PBS, and cut the tissue into approximately 1mm3 size;

2. Add 4 mL of enzyme digestion solution (0.1% and 0.1% type I collagenase) to the tissue block, suspend for 10 seconds, digest at 37 ℃ for 10 minutes, then use a dropper to prepare a single-cell suspension, naturally precipitate and collect the supernatant, terminate digestion with 10% FBS medium, and place at 4 ℃;

3. Add 3-4mL of enzyme digestion solution to the remaining tissue, suspend for 10 seconds, digest at 37 ℃ for 10 minutes, collect the supernatant according to the above method, terminate digestion, and place at 4 ℃. Repeat this step 2-3 times until the tissue is digested;

4. Filter the cell digestion solution through a 200 mesh stainless steel sieve, centrifuge at 1200r/min for 10 minutes, discard the supernatant, and suspend the precipitated cells in DMEM/F12 medium containing 10% FBS. Inoculate the cells into a 25cm2 culture bottle and incubate in a 37 ℃, 5% CO2 incubator;

5. After 1 hour of differential adhesion, aspirate the culture medium and inoculate it into a 6-well plate as needed for further cultivation;2、 Immunofluorescence identification:

1. When the atrial myocytes grow to 80% fusion, discard the culture medium and wash the cells twice with warm PBS for 10 minutes each time. Then fix the cells with 4% paraformaldehyde at room temperature for 15 minutes;

2. Wash the cells twice with PBS for 10 minutes each time, and then permeate the membrane with 0.1% Triton X-100 at 4 ℃ for 15 minutes;

3. Wash the cells twice with PBS for 10 minutes each time, and then block the cells with 4% BSA at room temperature for 30 minutes;

4. Dilute the alpha actin primary antibody in a ratio of 1:100, and then incubate the cells overnight at 4 ℃ in a refrigerator;

5. Wash the cells with PBS three times, each time for 10 minutes. Dilute the secondary antibody against alpha actin in a ratio of 1:150 and place it at 37 ℃ for 1 hour;

6. Wash with PBS three times for 10 minutes each time, observe the image under an inverted fluorescence microscope and take photos.

Cell Introduction:

The sclera is the outer layer of the eyeball wall, composed of dense collagen and elastic fibers, with a tough and opaque structure. The anterior edge of the sclera is connected to the corneal edge, and the posterior edge continues with the dura mater sheath of the optic nerve.

The sclera is divided from outer to inner into: upper layer of sclera; Main quality layer; Subcapsular layer. The upper layer is composed of epithelial cells.

Cell characteristics:

1） The organization is derived from normal eye tissue of experimental animals.

2)Cell identification: broad-spectrum keratin(PCK)Fluorescence staining is positive.

3)After identification, the purity of the cells was found to be higher than90%.

4)does not containHIV-1TheHBVTheHCVMycoplasma, yeast, and.

5)Cell growth mode: epithelioid, irregular cells, adherent culture.

Recommended culture medium:

We recommend usingDelfprimary culture epithelium Cell culture system As a culture medium for in vitro cultivation.

Notes:

1. After receiving the cells, first observe whether the cell bottle is intact and whether there is any leakage or turbidity in the culture medium. If any of the above phenomena occur, please contact us in a timely manner.

2. Carefully read the cell instructions and understand the relevant information about the cell, such as cell morphology, culture medium used, serum ratio, required cytokines, etc., to ensure consistent cell culture conditions. If there are any problems with the cell due to inconsistent culture conditions, the responsibility shall be borne by the customer.

3. Wipe the surface of the cell vial with 75% alcohol and observe the cell status under a microscope. Due to transportation issues, it is normal for some cells to form fragments due to temperature changes and severe collisions. After observing the cell state, disinfect the bottle wall with 75% alcohol and place the T25 bottle in a 37 ℃ incubator for 4-6 hours.

4. Adherent cells can be digested, suspended cells are directly mixed and collected, centrifuged at 900 rpm to 1000 rpm for 3 minutes, and the supernatant is discarded. Add 5 mL of PBS to resuspend the cells, centrifuge at 900 rpm to 1000 rpm for 3 minutes, resuspend the cells in fresh * medium, and inoculate them into new culture bottles or dishes for cultivation in an incubator.

5. Please ask the customer to use the same culture medium for cell culture under the same conditions.

6. It is recommended that customers take several photos of the cells in the first 3 days after receiving them, recording the cell status for communication and exchange with our technical department. Due to transportation reasons, some sensitive cells may experience instability. Please contact us promptly to inform us of the specific situation of the cells, so that our technical personnel can track and follow up until the problem is resolved.

7. This cell is for scientific research purposes only.

8. Note: The transport medium (infusion medium) cannot be used to culture cells anymore. Please use the newly prepared * medium according to the instructions for cell culture conditions to culture cells. After receiving the cells, it is recommended to subculture them at a ratio of 1:2.

9. Note: 1:2 passage refers to transferring one T25 bottle to two T25 bottles or two 6cm dishes. Not one T25 bottle to two 10cm dishes.

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