TRYPSIN-EDTA SOLUTION

Concentration: 0.05% (w/v) Trypsin (1:250) and 0.02% (w/v) EDTA (Versene)

Typical Protocol to Remove Adherent Cells from a Culture Surface:

1. Remove the culture medium from the culture vessel by aspiration and wash the monolayer with a Ca²⁺ and Mg²⁺ free salt solution to remove all traces of serum. Remove the salt solution by aspiration.
2. Dispense enough of the Trypsin-EDTA solution into the culture vessel to completely cover the monolayer of cells and place in a 37°C incubator for approximately 2 minutes.
3. Remove the Trypsin-EDTA solution by aspiration and return the closed culture vessel to the incubator. The coated cells are allowed to incubate until the cells detach from the surface.

NOTE: The time required to remove the cells from the culture surface is dependent on the cell type, population density, serum concentration in the growth medium, potency of the trypsin and time since the last subculture. Trypsin causes cellular damage and time of exposure should be kept to a minimum.

4. When the trypsinization process is complete, the cells will be in suspension and appear rounded.
5. It is advisable to add serum or medium containing serum to the cell suspension as soon as possible to inhibit further tryptic activity which may damage the cells.
6. Cells can be resuspended by gently pipetting the cell suspension to break up the cell clumps. Further dilution can be made, if required, for cell counts and/or subculturing.