Trypsin dissociation procedure
Trypsin is a serine protease, found in the digestive system of many vertebrates, where it hydrolyses proteins. It cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline. In cell culture, trypsin is used to break the focal adhesions that are anchoring the cell-culture dish as well as the cadherin-mediated cell-cell adhesion, so we can harvest individual cells. Trypsin's action mechanism is inhibited by serum proteins, calcium and magnesium; and in the presence of calcium, cadherins are resistant to trypsin. Therefore, most of the trypsin solutions also contain EDTA which acts as calcium chelation agent. Furthermore, we also need to wash our cells first using calcium-free and magnesium-free PBS. Finally, it is important to incubate the cells with trypsin no more than 2-5 minutes, since trypsin can be toxic to the cells after that time.
Take a look at this video to see how to perform all the necessary steps:
Normally, trypsin solution has an animal origin and must be stored at -20 °C and pre-warmed at 37 °C before use. Instead of trypsin, TrypLETM can also be used. Its components are animal- and human-free, stable at room temperature for at least 6 months, not inhibited by serum and do not require trypsin inactivators.