1. Prewarm sterile mediand any supplements to 37 °C in a water
bath. Don't leave media 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. Transfer a small aliquot of cells to an eppendorf tube. Count the cell concentration using a
5. 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.
6. Cap the flask loosely to allow air/CO2 diffusion. Incubate the flask
in the incubator.
7. 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. antibiotics).