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Q：What concentration of CO2 should be used when cultivating cells? 5% or 10%, or no impact at all?
Most culture media use HCO3-/CO32-/H+as a pH buffer system, and the content of NaHCO3 in the medium will determine the CO2 concentration that should be used during cell culture. When the NaHCO3 content in the culture medium is 3.7g per liter, 10% CO2 should be used for cell culture; When NaHCO3 is 1.5g per liter in the culture medium, cells should be cultured with 5% CO2.
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Q：Why does the purchased cell freezing tube have too few cells after thawing?
Researchers found that the number of cells after thawing was too low, mostly due to errors in the centrifugation process, resulting in physical damage to the cells and cell loss. It is recommended not to centrifuge the cells immediately after thawing. Wait for the cells to grow overnight before changing the culture medium.
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Q：How to avoid cell contamination?
The types of cell contamination can be divided into bacteria, yeast, mold, virus, and mycoplasma. The main reasons for cell contamination include improper aseptic operation techniques, poor operating room environment, contamination of experimental consumables, serum contamination, and contamination of cell sources. Strict aseptic operation techniques, clean environment, and high-quality cell sources are the best ways to avoid contamination.
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Q：What are the steps for cell cryopreservation?
Freezing method one: Place the freezing tube at 4 ° C for 30-60 minutes → (-20 ° C for 30 minutes *) → -80 ° C for 16-18 hours (or overnight) → Long term storage in liquid nitrogen tank vapor phase. Freezing method 2: Place the freezing tube in a programmable cooling machine that has been programmed to cool down 1-3 ° C to below -80 ° C per minute, and then store it in a liquid nitrogen tank vapor phase for long-term storage*- At 20 ° C, it should not exceed 1 hour to prevent excessive ice crystals from causing massive cell death. This step can also be skipped and placed directly in a -80 ° C freezer, but the survival rate will decrease.
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Q：What are the grades and sterile filtration methods of DMSO?
The DMSO grade used for cryopreservation must be Tissue culture grade, 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 away from light. 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. It must be prepared before use and pre cooled at 4 ° C.
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Q：What is the appropriate cell seeding density?
Inoculate according to the seeding density or dilution ratio on the basic data of the cell line. Too few cells or too many dilutions may also lead to slow cell growth.
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Q：What is the centrifugal speed for typical animal cells?
To recycle animal cells, the centrifugation rate is generally 300g (about 1000 rpm) for 5-10 minutes. Excessive rotation speed will cause cell death.
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Q：How should suspended cells be passaged?
Generally, it is only necessary to continuously add fresh culture medium to the original culture bottle and dilute the cell concentration. If there is too much culture medium, the mouth of the culture bottle can be slightly raised until it cannot accommodate it. When dividing the bottles, take out a portion of the culture medium containing cells and transfer it to another new culture bottle. Add fresh culture medium and dilute to the appropriate concentration. Repeat the above steps.
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Q：Do antibiotics need to be added to the culture medium?
Except for special screening systems, under normal cultivation conditions, no antibiotics should be added to the culture medium. However, in practical operation, sometimes 1% penicillin streptomycin is added to avoid bacterial contamination.
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