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 use.

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 hemocytometer.

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).