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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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Q：Can Mycoplasma contamination be visually recognized?
Yes, provided that your eyes have 20 million pixels and a 4000x zoom function. Generally speaking, cells contaminated with Mycoplasma will deteriorate in growth status, and it is difficult to distinguish with the naked eye whether it is Mycoplasma contamination or if you added a pinch of salt to your own culture of Kirido... The main way to distinguish Mycoplasma contamination is through PCR detection or staining detection. However, once contaminated, try to discard it as much as possible and check if your serum is safe.
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Q：How many days to change the culture medium?
Under normal circumstances, the culture medium tends to be red. If the cells are maintained at pH 6.5-6.6 and the culture medium turns yellow, it indicates that metabolic products have accumulated to a certain amount in the culture medium, and the cells will fall off and die, requiring replacement with fresh culture medium. Generally, when cells grow vigorously, they should be replaced every 1-2 days. When growth is slow, it can also be replaced every 3-4 days.
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Q：Why are many cells difficult to adhere to the wall after revival?
It may be due to poor state during cell cryopreservation or slow movement during recovery, leading to cell death. Remember to melt quickly and centrifuge to remove DMSO. If the non adherent cells still survive, they can be collected by centrifugation and re inoculated back into the original bottle, and the serum ratio can be adjusted to 15% to assist in adherent culture.
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Q：Why is the culture medium usually reddish?
Because phenol red is added as an indicator to the culture medium, indicating a pH range of 6-8, if the culture medium is acidic, it will turn yellow, and if it is alkaline, it will turn purple. It is necessary to change the medium in a timely manner and replace it with fresh culture medium to maintain a good growth environment for cells. Some special cells require phenol red free culture medium and need to be observed in a timely manner.
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Q：How to avoid the occurrence of sediment in serum?
Firstly, it is important to pay attention to the correct steps for thawing serum, and during the dissolution process, it is necessary to shake the serum evenly and slowly at regular intervals. We have found that sediment may increase in the following situations, which should be avoided as much as possible during use: (1) heat inactivated serum; (2) Cultivate serum at 37 ℃; (3) Repeated freeze-thaw cycles; (4) Gamma ray irradiation; (5) Long term storage at 2-8 ℃; (6) Leaving at room temperature for too long
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Q：What are the possible precipitates in serum?
Based on years of experimental research, fetal bovine serum and other serum used for cell culture may contain the following types of precipitates: (1) fibrin, which is a frequently occurring large precipitate that can reach 1-2mm and can be observed with the naked eye. Because serum is collected and rapidly processed at low temperatures, some fibrinogen (soluble precursors that form flocculent fibrin) remains in a dissolved state during the processing. After the final filtration and packaging, fibrin precipitates will coagulate in the bottle. (2) Calcium phosphate, which is also a common precipitate, usually causes serum turbidity and increases when cultured at 37 ℃. This sediment appears as small black dots under an inverted microscope, which appear to be active due to Brownian motion and are often mistaken for microbial contamination. (3) Cholesterol, fatty acid esters, and some proteins. They are also a common cause of sediment in serum.
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Q：Is it better to use a sealed lid or a breathable lid for the cultivation bottle?
Fine. Just when using a sealed cap culture bottle, rotate the cap to about 2/3 and do not tighten it completely. Reserve about 1/3 so that the cells can breathe; Some brands of sealed cap culture bottles have appropriate position indication marks on the bottle caps.
"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
