Media and Reagents

Serum Free Media for human ES cells on MEFs: can last for 7-10 days

Final Concentration Amount for 250ml Stock solution
80% DMEM-F12 200ml
20% KO Serum Replacer 50ml
1% Non-essential Amino Acids 2.5ml
1mM L-glutamine (see recipe) 2.5ml
0.1mM b-mercaptoethanol part of L-glutamine
4ng/ml bFGF (see recipe) 0.5ml

L-glutamine stock solution: Prepare on a per-use basis

  • 0.146g L-glutamine
  • 10ml Ca/Mg free PBS
  • 7 ul b-mercaptoethanol

bFGF Stock: Store aliquots at –20 or –70

  • 10ug vial of bFGF
  • 5ml 0.1% Fraction V BSA
  • Aliquot 0.5ml into sterile tubes

0.1% Fraction V BSA:

  • 0.1g Fraction V BSA
  • 100ml PBS with Ca and Mg
  • Filter Sterilize

Collagenase IV Split Media: This media lasts 2-3 weeks

  • Final concentration of 1mg/ml in DMEM-F12 media ie. 0.05g collagenase IV in 50ml

Cell Freezing Media

Cryopreservative Medium

Final Concentration Amount for 10ml Stock Solution
60% DMEM-F12 6ml
20% defined FBS 2ml
20% DMSO (do not filter sterilize) 2ml

Resuspension Medium

Final Concentration Amount for 10ml Stock Solution
80% DMEM-F12 8ml
20% defined FBS 2ml