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Q：Why does RPMI1640 medium change color?
When we usually use RPMI1640 culture medium, do we experience colors such as pink, purple, and rose red when half used or just opened for a period of time? What is the reason for this? In fact, when we purchase RPMI1640 culture medium, we usually see two components on the ingredient list: sodium bicarbonate and phenol red. It can be said that these two components are the main reasons for the discoloration of RPMI1640 culture medium. Let's first take a look at sodium bicarbonate. After opening the culture medium, sodium bicarbonate will decompose to produce CO2, causing the culture medium to become alkaline. At this point, we can loosen the culture bottle and place it in a CO2 incubator for a period of time, and the color of the culture medium will become normal. In addition, phenol red can also cause discoloration of RPMI1640 medium, and when the pH in the medium is too high, phenol red will also change color. Cells produce metabolites in the culture medium, which can also cause changes in acidity or alkalinity. Therefore, the fundamental reason for the discoloration of RPMI1640 culture medium is that the pH value of the medium has changed. When the pH value of the culture medium is greater than 7, the color tends to be purple; On the contrary, if the pH value is less than 7, the color tends to be yellow.
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Q：What is the difference between BrdU method and CCK-8 method?
The CCK-8 method reflects cell activity by detecting dehydrogenases in logarithmic growth stages, while the BrdU method detects DNA synthesis in cells. The CCK-8 method only requires the addition of reagents and can be detected by colorimetric method, while the BrdU method not only requires the addition of reagents but also the fixation of cells, which can be detected by luminescence method after antigen antibody reaction. The principles of the two are completely different. If only detecting cell proliferation, then the CCK-8 method is relatively simple.
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Q：Will bacteria react with CCK-8 reagent? (If you want to detect bacteria)
CCK-8 reagent hardly undergoes color reaction with bacteria. The bacterial defense system is very strong and does not undergo a reduction reaction.
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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!
"Heyuan Liji" is a joint venture between Heyuan Biotechnology (stock code: 688238) and Liji Biotechnology, specializing in the "Liji Biotechnology" and "Life-ilab" reagent brands
