Frequently Asked Questions - Answers to Reagent Product Issues-He Yuan Li Ji

Q：Is trypsin and collagenase acting the same?

To separate cells from each other, enzymes can be used to digest proteins that adhere between cells, such as collagen, laminin, fibronectin, elastin, etc. The enzymes used in the textbook are trypsin or collagenase, and the "or" here does not mean arbitrary selection because the target of action of these two enzymes is not the same.
Trypsin is a pancreatic product that can strongly digest soft tissues with less intercellular matrix, such as liver, kidney, thyroid, amniotic membrane, embryonic tissue, etc. It is ineffective for tissues rich in connective tissue, such as breast, synovium, uterus, fibrosarcoma, tumor tissue, etc. Moreover, if the digestion time is too long, it can also damage the respiratory enzymes and membrane proteins of cells, causing damage to cells. Therefore, after digestion, the cultured cells should be washed immediately.
Collagen enzyme is an enzyme extracted from bacteria, suitable for digesting fibrous tissue, epithelial tissue, and cancer tissue. It has a good digestion effect on collagen components in the intercellular matrix. Although the effect is slow, it has little effect on the cells themselves, so it can still achieve the purpose of dispersing cells.
Q：Why is the cell culture medium yellowish or purplish?

The yellowing of cell culture medium is due to excessive cell density, which produces a large amount of acidic metabolic waste. When the pH increases, it causes phenol red discoloration, indicating insufficient cellular nutrition. The purple color of cell culture medium is due to the gradual overflow of CO2 from the culture medium, causing the medium to become increasingly alkaline and turn purple.
Q：How to control the digestion time of pancreatic enzymes?

Under an inverted microscope, observe that the cells become significantly round, bright, and the intercellular space increases. Gently shake to see suspended cells, but stop when there is no large detachment (do not excessively digest or affect cell activity; it is recommended to observe and record the best time every 30 seconds for newly purchased cells).
Q：How can we improve the frequent contamination of primary cells during extraction?

Firstly, it is necessary to ensure that there is no bacterial contamination in the cell separation laboratory. If there is a problem, the laboratory needs to be disinfected, the incubator needs to be sterilized, and the availability of reagents and consumables needs to be checked. Secondly, attention should be paid to aseptic operations to ensure that reagents, scissors, tweezers, etc. are sterile. Thirdly, antibiotics such as bispecific and amphotericin can be added to the culture medium.
Q：What is the reason why primary cells that adhere to the wall are difficult to digest?

The solution to the prolonged cultivation of cells in a dish during the first generation can be to lower the enzyme concentration, extend the digestion time in a 37 ℃ incubator, or digest in stages.
Q：Can primary cells be cryopreserved? What is the reason for the poor survival rate after cryopreservation and resuscitation?

This depends on the type of cell. Some cell types, such as neurons, glial cells, and some slow growing epithelial cells, are not recommended for expansion culture and re freezing. Other cell types such as fibroblasts, astrocytes, renal mesangial cells, astrocytes, etc. can be expanded, cultured, and cryopreserved. However, it should be noted that the process of re freezing may lead to changes in cell growth performance. The low recovery rate after cryopreservation requires consideration of pre cryopreservation cell activity, cell volume, and cryopreservation system. The cryopreservation system refers to whether serum containing cryopreservation solution or one-step cryopreservation solution is used, and different cryopreservation methods correspond to different operating methods that need to be noted.
Q：Which generation is generally better for conducting experiments on primary cells?

Within 5 generations, 3 generations are best.
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.
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.
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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