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
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
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
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