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Making media with lots of supplements - How to add transferrin? (Jan/06/2009 )

Hi! I need to make media (Ham's F12, powdered) for a cell line (NL20) that requires lots of supplements:

Nonessential amino acids
Epidermal growth factor
4% FBS

I have never made media this complex before, and I was hoping someone could tell me the best way to do this.

ATCC suggests:
1. Combine all ingredients except EGF, up to 1 L.
2. Filter sterilize it.
3. Aseptically add the EGF.
4. Store it at 4oC.
5. When ready to use, aseptically add the 4% FBS and Pen/Strep.

However, I read that apo-transferrin (Sigma) is only stable in aqueous solutions for 5-10 days, but I also heard that the transferrin will be stable as long as there is FBS in the medium. I'm confused. So should I add this supplement (frozen aliquot) immediately before seeding the cells, or can I simply do as the ATCC suggests and make up a 1 L bottle of serum-free medium to store at 4oC.

Can someone please tell me how you make up media like this?


Nobody here uses tranferrin? Even if you use other supplements, can you please tell me how you make up your medium (especially if you start from powder....I'm new to using powdered media.)

Thanks so much if you can help!