Approximately 1 freezing vial per 25 cm2 flask.
3 vials from large size (75 cm2) culture flask.
Feeding Cells before freezing
- When cells are 90% confluent (~4 days), replace medium
- Draw out old medium, replace with 5 mL new (warm) medium.
- Make freezing medium (15% DMSO, 20% serum) in advance
- Remove medium from flask
- Rinse once with ~3 mL sterile PBS. (Squirt in, then suck out.)
- Add 10 – 15 drops trypsin (0.5 – 0.75 mL)
- Incubate (37C) 5 – 30 min
- Add 3 mL cold medium (regular)
- Pour into 15 mL centrifuge tube
- Optional – rinse with another 2 mL, to capture as many cells as possible
- Spin (~100-500xg) 5 – 10 min (until supernate is clear and pellet is hard)
- Pour off supernate (if pellet is solid – otherwise pipette it off)
- Resuspend in 1 mL freezing medium (pipette up and down)
- Transfer to freezing vial (should be slightly more than 1 mL)
- Put on ice
- Store overnight in -80C in "Mr. Frosty" (actually a VWR 414004-284 CryoCooler Freeze Controller)
- Put into liquid nitrogen.
Fill out the freezing log