Raft culture media (aka Green’s media) 3 parts DMEM (including glutamine or glutamax) 1 part Ham’s F12 5% FCS various supplements (not everybody uses the same supplements, some don’t use any!) The following are the supplements I have used. I doubt the necessity of the last three but have not got round to trying without them. Adenine (Sigma A8626), MW=135, final concentration=180mM,  working stock=45mM, Dilution factor=250. Dissolve in 0.05N HCl to 45mM. Store working aliquots at –20C. Insulin (Sigma I6634), MW=5733, final concentration=5mg/ml,  working stock=5mg/ml, Dilution factor=1000. Dissolve in acidified H2O (0.1ml glacial AcCOOH + 9.9ml H2O) I had to add more glacial acetic acid to make it dissolve. Store working aliquots at 4C. Hydrocortisone (Sigma H0135), MW=362, final concentration=400ng/ml,  working stock=1mg/ml, Dilution factor=2500. Follow instructions with vial (dissolve in EtOH then add equal volume of H2O) Store working aliquots at -20C. Transferrin (Sigma T2036), final concentration=5mg/ml,  working stock=500mg/ml, Dilution factor=100. Dissolve in media (DMEM or KGM) Store working aliquots at -20C. Pen and Strep Fungizone T3 (aka 3,3’,5-triiodo-L-thyronine) (Sigma T6397), MW=673, final concentration=0.2nM,  working stock=1mM, Dilution factor=7500. Dissolve in 1N NaOH to 1mg/ml. Dilute 1000-fold in 0.1N NaOH. Store working aliquots at -20C. Cholera toxin (Sigma), MW=87kD, final concentration=0.1nM,  working stock=50mg/ml, Dilution factor=5750. Careful! EGF (Sigma E1264), final concentration=5ng/ml,  working stock=100mg/ml, Dilution factor=20000. Dissolve in media (DMEM or KGM) Store working aliquots at -20C. (also, I have seen in some papers that people add ascorbic acid immediately before use. I have never tried this.) Example (for a little over 100ml raft medium) 70ml DMEM 25ml F12 5ml FCS Add the following to the FCS and sterile filter before adding to the media; 400ml of Adenine (45mM) 100ml of Insulin (5mg/ml) 40ml of Hydrocortisone (1mg/ml) 1ml of Transferrin (500mg/ml) 13.5ml of T3 (1mg/ml) 18ml of cholera toxin (50 mg/ml) 5ml of EGF (100 mg/ml) also; 200ml fungizone (250 mg/ml) 1ml Pen and Strep (100x strength (10000 units/ml and 5000 mg/ml))

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