Frequently Asked Questions - Answers to Reagent Product Issues-He Yuan Li Ji

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.
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.
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.
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.
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.
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.
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.
Q：Can we use serum with different culture conditions from the original one?

I can't. Serum is an extremely important source of nutrition in cell culture, so the type and quality of serum have a significant impact on cell growth. Serum from different species may vary in the amount or content of certain substances or molecules, and incorrect use of serum can often result in the inability of cells to survive.
Q：Can we use a culture medium with different cultivation conditions from the original one?

I can't. Each cell line has its own specific and adapted cell culture medium. If a culture medium different from the originally provided culture conditions is suddenly used, the cells may not be able to adapt immediately, resulting in poor cell state or even death.
Q：Should DMSO be removed immediately when thawing and culturing cells in cryovials?

Except for a few cells specifically marked as sensitive to DMSO, the vast majority of cell lines (including suspended cells) can be directly placed in 10-15ml of fresh culture medium after thawing, and the fresh culture medium can be replaced the next day to remove DMSO. This can avoid the problem of most thawed cells being unable to grow or adhere.

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