Common small problems of pancreatin digestion

In addition to the culture medium, trypsin is also an indispensable part of cell culture experiments. Although the basic techniques of cell culture experiments are similar and the culture conditions of each type of cell are different, this step is indispensable in the process of cell culture. -Trypsin digestion. What are the issues to be aware of when using pancreatin for cell digestion?

1. Why should trypsin be used during cell culture? What is the role of pancreatin?
Trypsin is a kind of protease. The restriction site is the carboxyl terminal peptide chain of Lys or Arg of the peptide chain. By degrading the protein at a specific position, the protein at the junction of the cell membrane and the culture dish wall is degraded, thereby causing The two are separated. At this time, the cells can become spherical due to the tension of their own internal cytoskeleton and the surface tension of the culture medium.

2. Using fetal bovine serum to culture cells, it is found that the serum can terminate the process when trypsinization is used. What is the reason?

The principle of serum termination is actually competitive inhibition, that is, the protein contained in excess bovine serum is combined with pancreatin, so that pancreatin loses the opportunity to digest cell proteins.

3. Why did the cells still form clumps after trypsin digestion? What could be the cause?

① Insufficient digestion time ② Insufficient pipetting strength ③ Insufficient concentration of trypsin digestion solution ④ Digestion and passage only after the cell density is too high ⑤ Improper storage of pancreatin, or use of expired pancreatin

4. What should be paid attention to when the pancreatin is aliquoted and frozen?

Try to divide the pancreatin into multiple bottles as much as possible, and observe the principle of using up about 3 times and filling only 2/3 of the volume. Because the volume will expand during cryopreservation, repeated freeze-thaw cycles using the same vial of pancreatin will reduce the digestion effect and increase the possibility of contamination.

5. How can we judge that the pancreatin digestion is complete?

Note: It is not that all the cells are distributed in a single circle with discrete intervals to indicate that the digestion is complete. Generally, the adherent cell layer is observed with naked eyes, as long as it can move, most of it moves like sand, and digestion should be stopped.

6. What kind of cell digestion is pancreatin suitable for? What is its digestive effect related to?

Trypsin is suitable for digesting soft tissues and passage cells with less intercellular substance, such as embryos, epithelium, liver, kidney and other tissues, but it is less effective for fibrous tissues and harder cancer tissues. The digestive effect of pancreatin is mainly related to pH value, temperature, concentration of pancreatin, size and hardness of tissue mass.

7. What are the effects of phenol red in pancreatin?

Phenol red is used as a pH indicator in the culture medium. It is red when it is neutral, yellow when it is acidic, and purple when it is alkaline. Studies have shown that phenol red can mimic the effects of steroid hormones (especially estrogen). To avoid sterol reactions, it is recommended to use phenol red-free medium when culturing cells, especially mammalian cells. Because phenol red interferes with the detection, when doing flow cytometry, a medium or pancreatin that does not contain phenol red will also be used.

8. Why can't the cells be digested with trypsin containing EDTA when testing for apoptosis?

Annexin V (Annexin) is a Ca2+-dependent protein. EDTA will chelate Ca2+ to affect Annexin V, which in turn affects the results. Therefore, trypsin without EDTA should be used. It is recommended to put it in an incubator for digestion, which can take a little longer. Observe the condition of the cells at any time to avoid excessive digestion; you can also use a cell scraper to scrape the cells, and then blow them evenly with a gun, which is simpler than digestion and can also avoid the damage caused by the use of pancreatin.