Trypsinizing cells

There are many procedures with which to trypsinize cells. All include washing the cell monolayer with TD, or in rare cases, with VE. This removes serum, which contains trypsin inhibitors, and lowers the concentration of both calcium and magnesium, both of which inhibit trypsinization. I like to rinse the cells with TD, add approximately 1 ml of straight trypsin, rock the dish, aspirate a majority of the trypsin, and then incubate the dish at 37 °C until the cells come off the dish. Some people put 1 ml of 5 fold diluted, with TD, trypsin on the dish and leave it on. Others do this using full-strength trypsin. Cells vary enormously in how fast they come off the dish. Some cells come off within one minute, some require 15 min. Cells should be trypsinized until they come off the dish, but not longer. Don't rush, but don't over do it. Cells that are extremely hard to dislodge with trypsin should be rinsed with VE prior to trypsinization and then trypsinized in the presence of VE. The EDTA promotes disaggregation. Unfortunately, EDTA is toxic to cells at high concentration--it deprives them of Zn++. It is essential to inactivate trypsin and to remove EDTA after you have gotten the cells off the dish. Trypsin inhibition is accomplished in part by trypsin inhibitors in the serum in the growth medium. Bovine serum is a rich source of trypsin inhibitors. The best, but most laborious way to reseed cells, is to dilute the suspended cells with medium containing serum and spin them down in the gray table top centrifuge--5/8 speed, 4 min. Then aspirate the supe, resuspend the cells in medium, and reseed them. A short cut that works with cells that are being seeded at a low cell density is simply to dilute the trypsinized suspension of cells with medium and seed the cells directly. If the cells are to be reseeded at a high cell density, the trypsin will not be diluted or inactivated sufficiently and the cells will not attach. This is not satisfactory when VE is used because EDTA is growth inhibitory even at low concentrations. It is also not satisfactory with chick cells, since they are grown in medium containing only 2% serum and cannot be seeded very sparsely.