Aspirate: Aspirate the media from the flask.

Rinse: Rinse with 5-10 mls of PBS (Ca++ & Mg++ -free).

Trypsinize: Add 1 ml of trypsin (in Hank's BSS) to flask, roll the flask around a bit to coat the growth surface.

Incubate:Put at 37°ree;C for a few minutes (~2-5 min?).

Whap: When surface looks ~ like clouded glass, whap the side of the flask with your hand to help dislodge the cells. The cells should come off in sheets and slide down the surface.

Quench: Add 3 ml media to flask ( total = 4 mls). The serum in the medium will inhibit the trypsin. May need to pipet the cells up and down to break up cell sheets.

Re-seed: Add 1 drop cells into a 25cm2 flask and 3 drops into another to re-seed the culture. Add 7-8 mls media. [--> ~1/15 dilution and 1/5 dilution from 75cm2 flask.].

Aliquot:Aliquot cells into new dishes, at 2:1, 10:1 or whatever split ratio you need. Add media.

Shirley Reynolds