1. Prewarm sterile media, Phophate Buffered Saline (PBS), Trypsin-EDTA, and any supplements to 37 °C in a water
bath. Don't leave media, PBS, Trypsin-EDTA, and any supplements in 37 °C longer than necessary.
2. Open tissue culture laminar flow hood and clean working area with 70% ethanol. The air flow
should have been turned on and the hood irradiated with UV light for at least 10 minutes. Turn off UV before
3. Wipe media bottles with 70% ethonal, and put them inside the hood.
4. Remove used media from a flask using an autoclaved pasture pipette
attached to the aspirator.
5. Wash off remaining media with 10-20 ml of PBS. Remove PBS.
6. Add a few ml Trypsin-EDTA to the cells. Use ~1 ml for a 25 cm2
flask. Incubate in the 37 °C incubator for a few minutes or until ~75% of cells have detached.
Monitor the extent of detachment using a microscope.
7. Add 5-10 ml of media containing serum, transfer the cells to a 15 ml centrifuge tube.
8. Centrifuge the cells at 2000 rpm for 5 minutes using a bench-top centrifuge.
9. Remove the supernatant.
10. Resuspend cells in 5 ml medium.
11. Transfer a small aliquot to an eppendorf tube. Count the cell concentration using a
12. Depending on cell concentrations, sub-culture the cells 1:2 to 1:10. Using
~5 ml of media per 25 cm2 flask. Scale up for bigger flasks.
13. Cap the flask loosely to allow air/CO2 diffusion. Incubate the flask
in the incubator.
14. Clean up the work area with 70% ethanol. And put all media/supplements back to 4 °C (e.g., media) or -20 °C (e.g. Trypsin-EDTA).