FOR Chinese Hamster Ovary Cells (ATCC 9606-CRL)

Gather the following items and place in the biological safety cabinet:

• Automatic pipetor and 25ml and 2ml pipette tips
• Micropipetor and pipette tips
• Bunsen burner and lighter

• Close biological safety cabinet door.

1. Turn on the uv light in the biological safety cabinet for 15min.
2. Defrost presterilized Fetal Bovine Serum (FBS) by placing plastic bag in cool water.
3. Spray the outside of the following with 70% ethanol and place in the biological safety cabinet:
   • 4 sterile Bellco spinner flasks: 2 are 500ml and 2 are 50ml
   • 1 sterile 100ml bottle to save excess media
   • 2 (500ml) bottles of presterilized Ham's F12 Medium
   • 1 (100ml) bottle of presterilized Fetal Bovine Serum (FBS)
4. Sterilely remove 45ml of Ham's F12 Medium from a 500ml bottle of Ham's F12 and place in a sterile 50ml sterile spinner flask.
5. Repeat with the second 50ml sterile spinner flask.
6. Sterilely remove 5ml of Ham's F12 Medium and place in the sterile 100ml bottle for excess media. (The bottle of Ham's F12 Medium will now contain 405ml.)
7. Sterilely pour the remaining 405ml of Ham's F12 Medium into one of the 500ml sterile Bellco spinner flasks.
8. Open second bottle of Ham's F12, remove 95ml, and dispense as excess media into the 100ml sterile bottle.
9. Sterilely pour the remaining 405ml of Ham's F12 Medium into the second 500ml sterile Bellco spinner flask.
10. Sterilely remove 45ml of FBS and dispense into one of the 500ml spinner flasks.
11. Repeat with second flask. (This will bring the total volume in each flask to 450ml, with 10% of the volume comprised of FBS.)
12. Sterilely remove 5ml of FBS and add to one of the 50ml spinner flasks.
13. Repeat the process with the second sterile Bellco spinner flask.
14. Proof all Spinner flasks containing CHO cell media in the CO2 incubator with stirring on the Bellco magnetic stirrer.
15. Purge the incubator with 5% CO2.