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Q：Should dual antibodies (penicillin&streptomycin) be added to the culture medium?
Dual antibodies are not necessary for cell growth. We do not routinely use antibiotics when cultivating cells, as continuous use of antibiotics can promote the development of antibiotic resistant cell lines, leading to persistent mild contamination. Once antibiotics are removed from the culture medium, this mild contamination will eventually develop into large-scale contamination. The specific situation can be decided whether to use bispecific antibodies based on the environment of one's own laboratory.
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Q：Why do we need to conduct cell passaging culture?
When the proliferation of cultured cells reaches a certain density, density inhibition will occur, manifested as a gradual slowdown or even cessation of cell growth and division speed. Adherent cells will grow into a dense monolayer in the culture bottle (suspended cells will fill the entire volume of culture medium), covering the bottom of the culture bottle. If not packaged and cultured in a timely manner, i.e., subculturing, the cells will gradually deteriorate and die. Transferring the cultured cells from one container to another at an appropriate ratio to expand the culture is called subculturing.
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Q：What culture medium can omit the addition of phenol red?
Phenol red is used as an indicator of pH value in culture media: it is red when neutral, yellow when acidic, and purple when alkaline. Research has shown that phenol red can mimic the effects of steroid hormones, especially estrogen. To avoid steroid reactions, it is not necessary to add when culturing cells, especially mammalian cells.
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Q：Is L-glutamine important in cell culture? Is it unstable in solution?
L-glutamine is important in cell culture. After removing the amino group, L-glutamine can serve as an energy source for cultured cells, participate in protein synthesis and nucleic acid metabolism. L-glutamine degrades in solution after a period of time, but the exact degradation rate has not been conclusively determined. The degradation of L-glutamine leads to the formation of ammonia, which is toxic to some cells.
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Q：Possible reasons for poor cell death or survival rate of purchased cells?
Researchers often experience poor survival rates during cell culture, which can be attributed to incorrect use of culture media or poor quality of culture media. Incorrect use of serum or poor quality of serum. Unfreezing process error. After thawing the frozen cells, wash the cells and centrifuge them. Suspended cells are mistaken for dead cells. Incorrect use of cultivation temperature. The cells were placed at -80 ℃ for too long.
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Q：What are the methods for cryopreservation of cells?
Freezing preservation method 1: Place the freezing tube at 4 ℃ for 30-60 minutes → (-20 ℃ for 30 minutes *) → -80 ℃ for 16-18 hours (or overnight) → store in a liquid nitrogen tank vapor phase for long-term storage. Cryopreservation method 2: Place the freezing tube in a programmable cooling machine that has been programmed to cool down 1-3 ℃ per minute to below -80 ℃, and then store it in a liquid nitrogen tank vapor phase for long-term storage*- At 20 ℃, it should not exceed 1 hour to prevent excessive ice crystals from causing massive cell death. This step can also be skipped and placed in a -80 ℃ freezer, but the survival rate is slightly reduced.
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Q：What are the grades of DMSO and the method of sterile filtration?
The DMSO grade used for cryopreservation must be tissue culture grade DMSO (such as Sigma D2650), which is already in a sterile state. After the first bottle opening, a small amount should be immediately divided into sterile test tubes and stored at 4 ℃ to avoid repeated freezing and thawing that may cause DMSO cracking and release harmful substances, and to reduce the chance of contamination. To filter DMSO, a DMSO resistant Nylon material filter membrane must be used.
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Q：What are the components of cell freezing culture medium?
The most commonly used cryoculture medium for animal cell cryopreservation is a mixture of 5-10% DMSO (dimethyl sulfoxide) and 90-95% fresh culture medium used for cell growth. Attention: Due to the release of a large amount of heat energy when DMSO is diluted, DMSO cannot be directly added to cell fluid and must be prepared before use.
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Q：What speed should be used to centrifuge general animal cells?
Recycling animal cells, the centrifugation rate is generally 300xg (about 1000 rpm) for 5-10 minutes. Excessive rotation speed will cause cell death.
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Q：What is the concentration of trypsin EDTA used during the subculture of adherent cells? How should it be handled?
The commonly used concentration of trypsin EDTA is 0.05% trypsin 0.53 mM EDTA Na4. After the first bottle opening, a small amount should be immediately divided into sterile test tubes and stored at -20 ℃ to avoid repeated freezing and thawing, which can reduce the activity of trypsin and reduce the chance of contamination.
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