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Q：How to perform gradient replacement of serum?
The replacement method is as follows: during the complete culture medium preparation process, mix 25% new serum with 75% original serum and culture for 1-2 passages; Then mix 50% new serum and 50% original serum for subculture; Then mix 75% new serum and 25% original serum for subculture; Finally, the cells were cultured and passaged entirely using new serum.
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Q：Is thermal inactivation of serum necessary?
Non essential serum does not require heat inactivation, while a few complement sensitive cell experiments, such as certain immunological experiments or adenovirus packaging, may require heat inactivation of serum as appropriate.
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Q：How to determine serum contamination?
(1) Check and determine if the outer packaging is damaged; (2) Inoculate the serum onto a blood lipid plate and culture it to see if there is colony formation; (3) It can be determined by the results of quality testing kits, such as mycoplasma testing kits. Serum contamination mainly includes bacterial contamination, fungal contamination, and mycoplasma contamination.
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Q：Is the small black dot in the serum a "black glue bug"?
The temperature difference during the serum thawing process is too large, and precipitation is more likely to occur after repeated freezing and thawing or thermal inactivation treatment. Some of these sediments appear as small black spots with Brownian motion when observed under a microscope, and there is a popular saying in the industry that they are contaminated by "black glue worms". Xiao L recommends Li Ji's black glue insect remover Cat: AC16L121
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Q：How to deal with flocculent sediment found after serum thawing?
The temperature difference during the serum thawing process causes lipoprotein denaturation and fibrin precipitation. The precipitation itself does not affect the quality of the serum, nor does it affect the experimental results. But if precipitation must be treated, it can be removed by centrifugation, such as centrifugation at 500-1000 × g for 5-10 minutes.
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Q：How to perform programmed thawing of serum?
Programmed thawing of serum can ensure the stability of serum product quality and prevent excessive protein precipitation due to large temperature differences. The programmed thawing method is to thaw the frozen serum in a 4 ℃ freezer before the experiment begins, and shake it evenly every hour during the thawing process to make the temperature and composition uniform, reduce precipitation, until it is completely thawed, and then transfer it to room temperature for use.
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Q：What are the conditions and methods for long-term storage of serum?
If serum needs to be stored for a long time, it must be stored in a refrigerator at -10 ℃ or lower, and it is recommended to store it in a refrigerator at -20 ℃. Research has shown that serum stored at -80 ℃ does not undergo significant changes in performance. However, due to the large temperature difference during thawing, more sediment may precipitate, resulting in a cluttered background. Therefore, long-term storage of serum at -80 ℃ is not recommended. If serum is frequently used in the near future, we recommend not storing it at 4 ℃ for more than one month. If it cannot be used in a complete bottle at once, it is recommended to pack and store it separately, and avoid repeated freezing and thawing.
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Q：Quick sealing solution works well, but if left for too long, it will settle and shake again?
It's Tween20. If left at room temperature for a long time, it will slowly oxidize and precipitate. Just shake it. Storing at 4 ℃ will be more stable and prevent precipitation.
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Q：Why do flocculent precipitates sometimes appear in serum? Do we need to remove it? How to remove it?
There are various reasons that may lead to the formation of flocculent precipitates, and one of the most common reasons is the aggregation of lipoproteins during the melting process. This phenomenon generally does not affect product quality. Centrifuge at 400 g for 5 minutes, take the supernatant, and then filter. Choose the appropriate pore size of the filter membrane according to the needs, and the most commonly used is the 0.22 um PES material filter membrane. To avoid membrane blockage, it is recommended to centrifuge and add the supernatant to the culture medium for filtration instead of directly filtering the serum.
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Q：Which is better in quality between fetal bovine serum and newborn bovine serum?
Fetal bovine serum is of the highest quality because it has not yet been exposed to the outside world, and contains the least harmful components such as antibodies and complement to cells; Bovine serum is the most commonly used natural culture medium in cell culture, containing abundant nutrients necessary for cell growth. It is commonly used for in vitro culture of animal cells and has extremely important functions.
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