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1. Newly bought or donated cells
1. How to deal with newly bought or gifted cells?
Regardless of the cells you buy or the cells you have given, you should do these things quickly when you get the hand: check whether the bottle is broken; whether the medium leaks, whether it is turbid; whether there is mycoplasma contamination; rapid expansion and cryopreservation.
2. Can I use a medium different from the original culture conditions?
No. Each cell strain has its own specific and adapted cell culture medium. If the medium is used differently and the culture conditions are different, the cells are mostly unable to adapt immediately, and the cells cannot survive.
3. Possible reasons for poor cell death or survival?
Common causes may be: incorrect use of the medium or poor quality of the medium; poor serum use or poor quality of the serum; thawing process error; thawing of frozen cells, washing cells and centrifugation; suspension cells mistaken for dead cells; culture temperature Incorrect use; incubator gas concentration is not required; cells are placed at –80 °C for too long.
Second, resuscitation of frozen cells
4. The cork tube that has been received is broken, the bottle cap is cracked, or the cap is peeled off?
Cracks in the chilled tube cap, or the rupture of the bottle, may be caused by improper force when the operator grips the cryotube, causing the cryotube to be cracked. It is recommended to use a hemostatic forceps to carefully grasp. In addition, the loose tube cap is loose or loose, which is a physical phenomenon due to thermal expansion and contraction. The cryotube may cause cell contamination. Therefore, when the cryotube is placed and removed, the cryotube should be immediately removed. Twist once.
5. How should the freezing tube be thawed?
When the chilled tube is removed from the liquid nitrogen tank, it must be safe to wear. Wear safety glasses and gloves to prevent the freezing tube from bursting. Immediately after taking out the cryotube, it should be quickly thawed in a 37 °C water bath environment, and gently shake the cryotube to melt it in 1 minute to avoid damage caused by slow infiltration of water into the cells to form intracellular recrystallization. And pay attention to the water surface can not exceed the edge of the frozen tube cover, otherwise it is prone to pollution.
6. When the cell cryotube is thawed, should DMSO be removed immediately?
Most laboratories now use broth to remove DMSO after thawing the cells, but some researchers believe that except for a few cells that are particularly sensitive to DMSO, most cell lines (including suspended cells), after thawing, It can be directly placed in a culture flask containing 10~15 ml of fresh medium, and the fresh medium should be replaced every other day to remove DMSO. This avoids the problem that most cells cannot grow or attach after thawing.
Third, cell culture fluid
7. How to choose the right type of medium?
Some examples of more classic media are cited:
8. How to choose a special cell line medium?
There is no fixed culture condition for culturing a certain type of cell. Cells cultured in MEM are likely to grow equally well in DMEM or M199. In conclusion, MEM is used for adherent cell culture, and RPMI-1640 is a good start for suspension cell culture. AIM V (12005) medium (SFM) should be selected for serum-free culture for various purposes.
9. What medium can be omitted to add phenol red?
Phenol red is used as an indicator of pH in the medium: red in neutral, yellow in acidity, and purple in alkaline. Studies have shown that phenol red can mimic the effects of sterols (especially estrogens). In order to avoid sterol reaction, cultured cells, especially mammalian cells, are cultured without phenol red. Due to phenol red interference detection, some researchers did not use phenol red-containing medium for flow cytometry.
10. What should I pay attention to when preparing and storing cell culture media?
After the cell culture medium is prepared, it should be taken in a culture flask and placed in a 37 ° C incubator for 24 to 48 hours. The culture solution has been tested for contamination before it can be used for experiments. The serum required to configure the medium is to be qualified and stable. After a batch test has a good effect, more serum of the same lot can be purchased at one time, so that the experimental conditions are stable, and it is easier to adjust the pH value during the dosing. It is advisable to use the amount of liquid for two weeks or so. Do not mix too much at one time. First, the loss of nutrients in the short-term use may require supplementation. Second, the amount is large and easy to cause pollution.
10. Why is the medium stored in a 4 °C refrigerator and the color is dark red?
The medium is stored in a refrigerator at 4 °C, and the CO2 in the medium will gradually overflow, causing the medium to become more alkaline, and the color of the acid-base indicator (usually phenol red) in the medium will be alkaline. Increased and more dark red. As a result of the alkalinity of the medium, cell growth arrest or death will result. If the medium is alkaline, the CO2 can be filtered through sterile filtration to adjust the pH.
12. When is serum-free medium used?
Serum-free medium should be selected for the isolation of cells using cultured cells to prepare cell growth factors, monoclonal antibodies, and the like.
13. How to choose the right serum type?
14. Can I use different serum types than the original culture conditions?
No. Serum is an extremely important source of nutrients in cell culture, so the type and quality of serum can have a significant impact on cell growth. Serum from different species varies in the amount or content of some substances or molecules, and errors in serum use often cause cells to fail to survive.
15. Is serum inactivation necessary?
This issue is now controversial. Some people claim that expensive serum contains precious substances such as growth factors, vitamins, and amino acids, and that heat treatment at temperatures above 50 °C for 30 minutes will bring about growth factors, amino acids and other components in serum. Negative impact. Nevertheless, heat inactivation of serum in most laboratories is performed routinely, especially in insect cell and embryonic stem cell culture.
16. Is there any antibiotic added to the medium?
Except in the special screening system, no antibiotics should be added to the medium under normal culture conditions.
17. When will the medium be changed?
Depending on the cell growth density, or according to the replacement time on the basic data of the cell line, the medium can be changed on time.
Fourth, cell passage
18. What are the methods of cell passage?
Different methods are used depending on the cells. Adherently grown cells are passaged by digestion; some adherently grown cells can be passaged by direct pipetting; cells that are suspended and grown can be passaged by direct blown or centrifugal separation, or the supernatant is removed by natural sedimentation, and then blown. .
19. What should be paid attention to in the first pass of primary culture?
Before the cells grow enough to cover most of the surface of the bottom wall of the bottle, do not rush to pass the passage; in the primary culture, the cells are mostly mixed, and different cells have different digestion time, so it should be observed and treated according to the needs, and according to different The cells are required to isolate and purify the cells for the tolerance time of trypsin; the action of the cells should be light and light to minimize the damage to the cells; the number of cells inoculated during the first passage should be increased, so that the cells can adapt to the new environment as soon as possible. Cell survival and value-adding; tissue pieces that fall off with digestion and separation can also be introduced into new culture flasks.
20. What is the purpose of adding EDTA when using trypsin? What should I do?
The effect of EDTA is more moderate than that of trypsin. The main function is to absorb Ca2+ and Mg2+ from the living environment of tissues. These ions are important factors for maintaining tissue integrity. However, EDTA alone can not completely disperse the cells, so it is often mixed with trypsin in different proportions, and the effect is better. The usual ratio is 1:1 or 2 parts EDTA: 1 part trypsin. The working solution concentration of EDTA is 0.02%, and it is configured with BSS containing no Ca2+ or Mg2+. If EDTA is used, the culture solution should be added after buffering the digestive solution before terminating digestion. Immediately after the first opening of the bottle, it should be dispensed in a small amount in a sterile test tube and stored at –20 °C. Avoid repeated freezing and thawing to reduce the activity of trypsin and reduce the chance of contamination.
21. How much speed should the centrifugal rate be to centrifuge the general animal cells?
To recover animal cells, the centrifugation rate is generally 800~1000 rpm, 5~10 minutes, and the excessive rotation speed will cause cell death.
22. What is the density of cell seeding?
Inoculation can be carried out according to the seeding density on the basic data of the cell line or the ratio of the dilution plate. Too few cells or too much dilution is one of the important reasons why cells cannot grow.
23. Possible causes of cell cross-contamination?
When a variety of cell lines are maintained for passage, the equipment and solutions required for each cell line are not strictly separated, and one cell is often contaminated by another.
Five, cell cryopreservation
24. Why should I add DMSO to the frozen medium?
Because the cells are directly frozen without any protective agent, the water inside and outside the cells will quickly form ice crystals. The formation of ice crystals will cause cell damage and cause cell death. At present, glycerin and DMSO are often used as protective agents. These two kinds of cells have no obvious toxicity, small molecular weight, large solubility, easy to penetrate cells, can lower the freezing point and improve the permeability of the envelope to water.
25. What is the grade of DMSO and the way to sterile filtration?
The DMSO grade used for cryopreservation must be Tissue culture grade DMSO, which is itself sterile. Immediately after the first opening, it should be dispensed in a small amount in a sterile test tube and stored at 4 °C to avoid repeated freezing and thawing. The cleavage of DMSO releases harmful substances and reduces the chance of contamination. To filter DMSO, a DMSO-resistant Nylon filter is required.
26. How much cell concentration should there be in the cryotube when cryopreserved?
The number of cells in the cryotube is generally 1x106 cells/ml vial, and the fusion tumor cells are preferably 5x106 cells/ml vial.
27. How to cryopreserve cells?
Cryopreservation method 1: Freezing tube placed at 4 °C for 30~60 minutes → -20 °C for 30 minutes → -80 °C for 16~18 hours (or overnight) → Liquid nitrogen tank vapor phase Long-term storage.
Cryopreservation method 2: The cryotube is placed in a programmable cooling machine with a programmed procedure to reduce the temperature by 1~3 °C to below -80 °C, and then stored in the vapor phase of the liquid nitrogen tank for long-term storage. Do not exceed 1 hour at –20 °C to prevent excessive ice crystals and cause a large number of cell deaths. You can skip this step and put it directly into the –80 °C refrigerator, the survival rate is slightly lower.
28. Will it be wasted if the cells are frozen too much?
Each cell line should have sufficient frozen storage to prevent the extinction of cell lines due to factors such as cell contamination, and the state of the cells in each batch is different. Several batches of better state should be selected. Save the species. In addition, finite cell lines such as diploid cells should be frozen if they are not used for a long time to avoid passage, causing cell aging or alteration.
Well, the notes on cell culture are discussed here today. Do you have anything to add? Come and contact me!
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Things to watch out for when culturing cells
Things to watch out for when culturing cells <br> Cell culture plays an important role in life science research. Many of the results of the experiment can be described as the cell culture of the adult, and the cell culture of the defeat. However, the cell abuses me thousands of times, I wait for the cell as first love! Researchers are not so easily defeated. There are countless posts on cell culture on various platforms, and hundreds of families argue. This article collects and organizes related books and industry platform notes on cell culture, and hopes to help everyone.