Chicken embryo fibroblasts (UMNSAH/DF-1)
Cell introduction
The cell is a chicken cell line derived from a 10-day-old ELL-0 egg, spontaneously immortalized. Primary chicken embryo fibroblasts were isolated and cultured in culture medium; passaged until senescence; centrifuged during senescence to maintain cell culture at 30% to 60% full; non-senescent clones were identified and passaged no less than 30 times. No clonal proliferation was observed on soft agar, indicating that these cells were immortalized without transformation. The cell serves as a substrate for viral proliferation, recombinant protein expression, and recombinant virus production.
Cell characteristics
1) Source: Chicken embryo
2) Morphology: fibroblasts, adherent growth
3) Content: >1x106 /mL
4) Pollution: Negative detection of mycoplasma, bacteria, yeast and fungi
5) Specifications: T25 bottle or 1mL frozen tube packaging
Transportation and preservation: You can choose dry ice transport and send resuscitation of viable cells: (1) dry ice transport, immediately transferred to liquid nitrogen or -80 degree refrigerator frozen or directly resuscitated after receipt; (2) surviving cells, should be received after receipt Continue to grow, pass the passage to reach a good cell growth state, and then freeze. See the cell culture step for specific procedures.
Please take a photo after receiving the cells. If you find any pollution within 3 days, please take a photo and contact us.
Cell use: For research use only.
Treatment after receiving the cells:
1) After receiving the cells, please check for leaks. If the liquid leaks, please send us a photo.
2) Please confirm the cell growth state under the microscope, sterilize the bottle wall with alcohol and place the T25 bottle at 37 °C for about 2-3 hours.
3) Discard the medium in the T25 bottle and add 6 ml of complete medium.
4) If the cells are over 90%, please pass the cells in time.
The company's cell culture operating procedures for reference
one. Preparation of culture medium and culture cryopreservation conditions:
1) Prepare DMEM medium; high quality fetal bovine serum, 10%; double antibody 1%
2) Culture conditions: Gas phase: air, 95%; carbon dioxide, 5%. Temperature: 37 ° C, incubator humidity of 70% -80%.
3) Cryopreservation solution: 90% serum, 10% DMSO, ready to use.
two. Cell processing:
1. Resuscitation cells: The frozen tube containing 1 mL of the cell suspension was quickly thawed in a 37 ° C water bath, and the tube was added to 4 mL of the medium to mix well. Centrifuge at 800-1000 RPM for 4-5 minutes, discard the supernatant, add 1-2 mL of medium, and mix well. All cell suspensions were then added to T25 flasks and supplemented with medium to 6 ml.
2. Cell passage: If the cell density reaches 80%-90%, subculture can be carried out.
1) Discard the culture supernatant and wash the cells 1-2 times with PBS containing no calcium or magnesium ions.
2) Add 1ml of digestive juice (0.25% Trypsin-0.53mM EDTA) to the culture flask, digest it in the incubator at 37 °C for 1-2 minutes, then observe the cell digestion under the microscope, if the cells are mostly rounded and fall off Quickly take it back to the console, tap a few flasks and add a small amount of complete medium to stop the digestion.
3) Gently mix and aspirate, centrifuge at 1000 RPM for 5 minutes, discard the supernatant, add 1-2 mL of the culture solution, and mix well.
4) Dispense the cell suspension into a new dish containing 6 ml of medium or bottle in a ratio of 1:2 to 1:5.
3. Cryopreservation of cells: When the cells are in good growth state, the cells can be frozen. The following T25 bottles are of the same type;
1) When the cells are frozen, the medium is discarded, and after washing with PBS, 1 ml of trypsin is added, and after the cells are rounded off, the digestion is terminated by adding 1 ml of complete medium, which can be counted using a hemocytometer.
2) Remove the supernatant by centrifugation at 4 min 1000 rpm. Add 1ml serum to resuspend the cells, add serum and DMSO according to the number of cells, mix gently, the final concentration of DMSO is 10%, the cell density is 1-2xE6/ml, and each cell is frozen and stored in 1ml cell suspension, pay attention to cryopreservation. The tube is well marked.
3) Place the cryotube in the program cooling box, put it into the -80 degree refrigerator, and transfer it to liquid nitrogen for storage at least 2 hours later. Record the location of the cryotube for the next time.
Precautions:
1. After receiving the cells, if you find that the dry ice has evaporated, the cap of the cryotube is detached, damaged, and the cells are contaminated, please contact us immediately.
2. All animal cells are considered to be potentially biohazardous and must be operated in a secondary biosafety station. Please pay attention to protection. All waste liquids and vessels that have been exposed to the cells need to be sterilized before disposal.
[Sample requirements]
The collected nasopharyngeal swab samples should be transported at 2°C to 8°C and sent for inspection immediately, and the sample delivery and storage time should not exceed 48 hours.
[Testing method]
1. Before sampling, mark the relevant sample information on the label of the sampling tube.
2. According to different sampling requirements, use a sampling swab to sample in the nasopharynx.
3. The specific sampling methods are as follows:
a) Nasal swab: Gently insert the swab head into the nasal palate, stay for a while and then slowly turn to exit. Wipe the other nostril with another swab, immerse the swab head in the sampling solution, and discard the tail.
b) Pharyngeal swab: Wipe bilateral pharyngeal tonsils and posterior pharyngeal wall with a swab, also immerse the swab head in the sampling solution, and discard the tail.
4. Quickly put the swab into the sampling tube.
5. Break the part of the sampling swab higher than the sampling tube, and tighten the tube cover.
6. Freshly collected clinical specimens should be transported to the laboratory within 48 hours at 2°C to 8°C.
[Explanation of test results]
After the sample is collected, the sampling solution turns slightly yellow, which will not affect the nucleic acid test result.
[Limitations of the test method]
1. For samples that are seriously contaminated due to improper storage after collection, the final test results will be affected.
2. If the sample is not stored at the specified temperature, the final test result will be affected.
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