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Q：How to determine bacterial contamination in cultured cells?
After overnight cultivation of cells contaminated with bacteria, the culture medium will significantly turn yellow, and bacterial sediment can be seen with the naked eye in the culture bottle. Shaking the culture medium will cause turbidity, and under a microscope, cells can be observed with fine sand covering the entire bottom of the culture bottle. The distinction between cell debris and contamination can be determined by overnight cultivation. The main components that do not become cloudy overnight are cell debris and metabolites, while those that become significantly yellow and cloudy overnight can be identified as contamination. Bacterial contamination of cells usually erupts to a visible state within 1-2 days, and bacterial contamination generally does not cross contaminate. Timely disposal of contaminated cells is sufficient.
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Q：Can Trizol be added after centrifugation of adherent cells after digestion? Does it have an impact?
Sure, resuspend with a small amount of PBs and extract with triazol, but the extraction should be fast and cannot be stored because the cells are living and their expression is affected by the external environment. If they have been stored for two hours, it is recommended to redo.
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Q：How to deal with pancreatic tissue RNA always sticking to the mortar during grinding? Is there any good way?
Immediately immerse the pancreatic tissue in liquid nitrogen after removal, then transfer it to a mortar (the mortar needs to be dried at 180 degrees for 4-8 hours to remove RNAase), continue grinding, add liquid nitrogen while grinding, and grind it into powder. At this point, it is important to pay attention to the replenishment of liquid nitrogen. If the tissue breaks down, there may be a situation where the tissue sticks to the mortar. Grind into powder and addTrizol，At this point, since Trizol will also freeze at low temperatures, continue grinding until Trizol dissolves and becomes clear. Then centrifuge and proceed with the following steps. You can also use a homogenizer, which is very cheap and simple. Of course, it needs to be processed with RNAase. Grind the tissue into a homogenate on ice.
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Q：What is the difference in the number of suspended cells and adherent cells?
Because of the difficulty of staining suspension cells, it is necessary to increase the number of cells and extend the time of culture. Staining of adherent cells is relatively easy, and if the number of cells is too large, sometimes the absorbance will exceed the reading of the microplate reader.
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Q：How many cells should be seeded in each well for a live cell test?
When standard 96-well plates were used, the minimum inoculum size of adherent cells was at least 1,000 cells per well (100 μl of medium) . The sensitivity for detecting white blood cells is relatively low, so a dose of no less than 2,500 cells per well (100 μl of medium) is recommended.
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Q：Can Cck-8 stain living cells?
No. Because the major component of CCK-8 is a water-soluble tetrazolium salt (WST-8) , and electrons in living cells are exchanged by the electron carrier PMS onto WST8 in the medium. CCK-8 can not stain cells because the resulting jazzans are also highly water-soluble.
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Q：How can the error due to CCK-8 residue on the gun head or hole wall be reduced?
CCK-8 reagent can be diluted with medium and mixed before adding sample.
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Q：Is it necessary to test whether the medium and CCK-8 react before the experiment?
If necessary, check it with a hole. Since there may be a redox substance in the medium, it is necessary to confirm that the medium reacts with CCK-8 before a formal experiment. The normal OD should be below 0.4.
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Q：CAN CCK-8 detect bacterial cells?
You can test for E. E. coli, but could not detect yeast cells. To 100 ΜL. Coli culture medium was added with 10 μl CCK-8 solution and cultured for 1-4 hours or overnight.
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Q：Storage conditions for CCK-8 reagent?
The storage condition of CCK-8 kit of Liji Biology: 4 ° C short-term storage, valid for 3 months; -20 ° C long-term storage, valid for 24 months. [ note ] : repeated freezing and thawing will result in higher background OD values.
"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
