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 use.

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 hemocytometer.

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).