1. When most cells are digested, just wash them with trypsin. After the trypsin is aspirated, the remaining trace amounts of pancreatic enzyme attached to the cell surface that are not calculated in volume are generally sufficient to digest the cells at 37 ° C for less than 2 min. For these cells, in principle, do not incubate the cells with trypsin, and continue to pass this way, which is very harmful to the cells. The simple procedure is to wash the PBS, wash it with trypsin, and digest it at 37 °C. More difficult to digest the cells (the washing method can not be digested for 5 minutes), such cells generally need to be incubated with a small amount of trypsin. 2. How can cells be digested? It is not a single circle in which the cells are distributed in a very discrete manner. It is generally digested. Generally, you can observe the adherent cell layer with the naked eye. As long as it can move, most of it is moving in the sand. It is already OK. Many people like to digest the cells or It is not necessary to blow into completely separated cells. Generally, it can move, indicating that the adhesion of cells to the culture substrate has disappeared, the adhesion between cells has disappeared, and the cells have been independently distributed (although there is no widely distributed discrete distribution). At this time, you should stop digesting. Don't wait until you see all the cells under the microscope are separated very well. The cells are completely in a single cell suspension, and then accumulate during the adherence process. This is a characteristic of adherent cultured cells, especially tumor cells. If it is inconsistent with the standard form, it may be caused by not digesting itself. However, if the digestion method is correct, it will still be distributed in pieces, or even completely blown into a single cell suspension. After adherence, it will still be distributed in pieces. This is the characteristic of cells. Because it was re-aggregated during the process of adhering. At this time, you are desperately trying to distribute it evenly, and your cells will become worse and worse for you. 3. The role of EDTA . Trypsin cleaves some of the proteins responsible for adhesion and attachment of ECM, while EDTA acts on the activity of Integrin by chelation of Ca ions, so the effect of EDTA is milder. Some people add some EDTA to the trypsin, or to treat cells that are particularly difficult to digest, and add more EDTA. Electronics Industrial Nitrile Gloves Electronics Industrial Nitrile Gloves,Pure Nitrile Exam Gloves,Waterproof Gloves For Work,Astm D6978 Examination Gloves Puyang Linshi Medical Supplies Co., Ltd. , https://www.linshimedical.com
4. Wash with PBS . Washing with PBS prior to digestion is a common procedure because the serum contains trypsin-inhibiting proteins. For some indigestible cells, PBS without Ca, Mg ions can be formulated because these ions also inhibit pancreatic enzyme activity. However, for most cells that can be digested with trypsin or EDTA solution, it is not necessary to formulate such a solution.