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Q：What is the difference in inoculation quantity between suspended cells and adherent cells?
Most suspended cells are more difficult to color and have lower O.D. values compared to adherent cells. Therefore, it is generally necessary to increase the number of cells inoculated or extend the culture time after adding CCK-8. The color development of adherent cells is relatively easy. If the number of cells is too large, the absorbance may sometimes exceed the reading of the enzyme-linked immunosorbent assay (ELISA) reader.
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Q：Is the sensitivity of CCK-8 the same for different cells?
The dehydrogenase activity and color rendering degree of different cells are different, so the sensitivity is also different.
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Q：What should be paid attention to in order to improve transfection efficiency?
1: The density of cells during transfection is 90% -95%. 2: During transfection, MEM serum-free medium should be used for nucleic acid and liposome dilutions. 3: You can choose to change the medium 4-6 hours after transfection.
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Q：Why do protective agents need to be added to cell cryopreservation?
Cell cryopreservation is a technique that places cells in a low-temperature environment to reduce cellular metabolism for long-term storage. When cells are frozen without any protective agents, the water inside and outside the cell quickly forms crystals, leading to a series of adverse reactions: firstly, some proteins denature due to increased local electrolyte concentration and pH changes, resulting in changes in the internal spatial structure of the cell. Secondly, the formation of ice crystals within cells and the denaturation of proteins and enzymes on the cell membrane system can limit the energy metabolism of cells. Once again, the lipid protein complexes on the cell membrane are prone to damage during freezing, leading to changes in membrane permeability and loss of cellular contents. Finally, as the freezing temperature decreases, the volume expansion of ice crystals causes damage to the spatial structure of DNA, leading to cell death. Adding cryoprotectants can effectively improve cell damage and death.
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Q：I have prepared too much cryoprotectant and don't want to waste it. Can I keep it for next use?
The prepared cryopreservation solution can be stored temporarily for one week at 4 ℃ and up to one month at -20 ℃. However, it is best to use it immediately and avoid repeated freezing and thawing.
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Q：After cell revival, only a very small number of cells adhere to the wall. Do we need to revive them?
Don't rush to throw it away yet! Serum and cytokines can be added, or fresh growth medium can be changed every 2-3 days. After 2-3 medium changes, most cells can show significant proliferation. At this time, normal passage operations can be followed. Of course, if the cell inventory is sufficient or there is an urgent need to conduct experiments, a new cell line can be revived~
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Q：Why was it that when I revived the cells, they were already attached to the wall, but when I went to see them the next day, I found that all the cells were dead?
It may be due to excessive digestion before cell cryopreservation, leading to cell apoptosis, so it is important to strictly control the digestion time when digesting cells!
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Q：Why are my revived cells not adhering to the wall at all?
It may be due to poor growth status before cell cryopreservation or slow movement during the recovery process, so it is necessary to select cells with good growth status and in logarithmic growth phase for cryopreservation, and the operation process should follow the principle of "slow freezing and fast thawing"!
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Q：Why is EDTA added to pancreatic enzymes?
Divalent ions can inhibit pancreatic enzyme activity, so adding EDTA can chelate divalent ions such as Ca and Mg. Be careful not to repeatedly freeze and thaw pancreatic enzymes!
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Q：What is L-glutamine used in the culture medium?
The main function of L-glutamine is to provide energy sources for cultured cells, participate in protein synthesis and nucleic acid metabolism. L-glutamine will degrade in solution for a period of time, but the exact degradation rate is indeed unclear. The degradation of L-glutamine leads to the formation of ammonia, which is toxic to some cells.
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