Trypsinization of Adherent Cells

Contributor: Suprya Jayadev
Date: August 8, 1991

1. Examine the falsk of cultured cells under the inverted microscope to check for contamination, cell density, etc.

2. Remove the old (spent) media.

3. Wash the monolayer with PBS (1 or 2 times). Let the PBS sit on the cells for at least 30 seconds to remove as much extracellular proteins as possible.

4. Remove the PBS and add a minimal volume (ie 0.9 ml for a T-75 flask) of 0.25% w/v trypsin solution which has been prewarmed to 37°C.

5. Let sit in incubator for ~3 minutes, then check flask under inverted microscope to make sure cells are the monolayer of cells is lifting off of the flask.

6. When a single cell suspension has been obtained, add ~10 times volume of complete media as trypsin solution.

7. Transfer the cell suspension to a sterile 15 ml conical centrifuge tube.

8. Spin the cells down at 1,200 rpm for 10 minutes in the tabletop centrifuge.

9. Discard the supernatant carefully, so as not to disturb the cell pellet.

10. Resuspend the cells in ~5 mls of fresh complete media and collect ~100 µl to count cell number.

11. Once cells/ml has been determined, seed cells into a fresh T-75 flask to get a final concentration of ~2 x 105 cells/ml.