Protocols
Required Media
Serum Free Media for Human ES Cells on MEFs
Final Concentation
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Amount for 250 ml
Stock Solution
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| 80% DMEM-F12 (Invitrogen) |
200 ml
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| 20% KO Serum Replacer (Invitrogen) |
50 ml
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| 1% Non-essential Amino Acids |
2.5 ml
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| 200 nM L-glutamine/βmercdaptoethanol stock (see recipe) |
1.25 ml
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| 4 ng/ml basic FGF (see below) |
0.5 ml
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Filter sterilize using a 0.22 µm filter into a bottle which never cam in contact with detergents. Media are stored at 4°
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3. Preparation of basic FGF stock solution
Dissolve 25 μg of basic FGF (R&D Systems) in 12.5 ml Ca/Mg-free PBS (Invitrogen) containing 0.1% BSA.
Assure pipette tip is moistened before dissolving basic FGF. Basic FGF is by its nature very sticky and will bind to a dry pipette tip. Improper handling of basic FGF will negativley affect the success of your hES culture.
After dissolving the basic FGF prepare 0.5 ml aliquots, which should be stored at –80˚C. Use one aliquot of 0.5 ml basic FGF solution per 250 ml media. This brings bFGF to a final concentration of 4 ng/ml and is sufficient to promote hES cell growth and prevent differentiation.
200 nM L-glutamine/b-mercaptoethanol stock solution
Add 7 μl β-mercaptoethanol (Sigma) to 5 ml 200 mM L-glutamine. Store the aliquots at -20˚C.