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: primary anti-rat α-striated muscle actin, secondary antibody FITC-labeled goat anti-mouse IgG, ethanol-acetone mixture (1:1).
Second, the experimental equipment
1. Petri dish
2, culture bottle
3, direct cut and eye scissors
4, ophthalmology
5, 200 mesh screen
6, glass dropper
7, beaker
8, 15ml centrifuge tube
Third, the experimental process <br>
The myocardial tissue was taken and washed 3 times in D-Hanks.
Cut to 1mm3+ digestive juice (PriCells)
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37 ° C water bath for 8 min, aspirate the supernatant suspension (non-myocardial cells)
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Remaining tissue plus digestive juice (PriCells) magnetic stirring at 37 ° C for 10 min, 60 r / min
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Upper suspension plus digestion stop solution (PriCells)
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The remaining tissues are further digested with digester (PriCells) and continue to digest 3-5 times.
Collect all suspensions through a 200 mesh screen
Collect filtrate 1000RPM/10min
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Remove the supernatant, collect the pellet, and resuspend in 2 ml of medium.
Staining count
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: 1-3 days old baby rats.
3. Material pretreatment: The obtained heart was placed in 1×PBS (pH 7.4) containing 1% P/S, the atrium was removed, and repeated washing was performed until there was no blood stain in the heart chamber, and the washing liquid was clarified.
4. Digestion: Cut the ventricular muscle into 1mm3 size tissue block with ophthalmology, add 10ml of digestive juice, and absorb the upper layer suspension after 8min in 37°C water bath. The remaining tissue is added to the digestive juice 10ml, 37°C water bath and stirred by magnetic stirrer. 10 min; aspirate the upper suspension to terminate the reaction; the remaining precipitate was digested with the digestive juice 3-5 times until the tissue block was completely digested.
5. Termination of digestion: The digestion reaction solution was added at a ratio of 1:1 to stop the enzyme reaction.
6. 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.
7. Culture cell density: The cell density was adjusted and inoculated into the culture flask at 5 × 105 cells/ml.
8. Culture: Place in a 37 ° C, 5% CO2 incubator.
V. Cell identification
1. Microscopic identification: The morphology of the cells became fusiform or irregularly flat at 12h after inoculation. After 24h, the spontaneous contraction of individual cells can be seen, and then the cells gradually spread out to form pseudo-foot, forming an irregular star. On days 3 to 4, the cells are intertwined into a network to form a cell monolayer or cell cluster.
2. Immunohistochemical identification: detection of α-striated muscle actin 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 antibodies: Dilute the antibody according to the dilution requirements, add the antibody, and incubate at 4 °C overnight.
6. Washing: Wash 3 times × 15 minutes in 1 × PBS (pH 7.4) and dry.
7. Labeled antibody: Add fluorescently labeled antibody and incubate for 1 h at 37 °C.
Washing: Wash 3 times × 15 minutes in 1 × PBS (pH 7.4) and allow to dry.
8. Cover: Seal the tablet.
9. Microscopic examination: Under the fluorescence microscope, the cytoplasm was stained with brownish yellow, indicating that the cells were positive for the expression of α-striated muscle actin.
Six, matters needing attention
1. Choose young rats born 1-3d.
2. Sterilize the rats before removing the heart.
3. Use multiple digestions until the tissue block is completely digested, reducing the damage of the digestive juice to the cells.
4. Since cardiomyocytes account for only 25% of total cells, differential adherence is performed before inoculation of cardiomyocytes, and most of the fibroblasts are removed.
5. The cardiomyocytes obtained from the initial isolation are prone to lose contractile activity in the physiological concentration of calcium ion solution. In order to maintain the contractile function of the cardiomyocytes, the 4°C calcium-free Hanks' buffer should be used to soak and rinse the myocardium during the separation process. organization.
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