Trypsin causes cells to come off of the plate.

Trypsin is an enzyme that cuts proteins. In cell culture, we use trypsin to gently remove our cells from the plastic growth surface. It works by cutting the proteins on the surface of the cell that adheres the cell to the plastic. Once the proteins are cut, the cells float off the surface and into the liquid media. Once in suspension, you can count your cells and replate at the right concentration.

To count the cells you will use a blue dye called trypan blue. You will add an equal amount of trypan blue to a small aliquot of your cells suspension and then observe those cells using the microscope to determine their concentration.

Trypan blue is able to selectively color dead cells blue. Only cells with compromised cell membranes are able to uptake the dye and become stained. Healthy, viable cells with healthy cell membranes will not uptake the dye and will remain clear. We only count the living cells as dead cells will not lead to further growth.

Using this method we are able to determine the number of living cells in order to subculture our cells at the correct concentration.

### Learning Objectives:

- 1.5 - Subculture cells.