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=180m M,  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=5m g/ml,  working stock=5mg/ml, Dilution factor=1000.

Dissolve in acidified H2 O (0.1ml glacial AcCOOH + 9.9ml H2 O) 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 H2 O) Store working aliquots at -20C.

Transferrin (Sigma T2036), final concentration=5m g/ml,  working stock=500m g/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=1m M, 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=50m g/ml, Dilution factor=5750.

Careful!

EGF (Sigma E1264), final concentration=5ng/ml,  working stock=100m g/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;

400m l of Adenine (45mM) 100m l of Insulin (5mg/ml) 40m l of Hydrocortisone (1mg/ml) 1ml of Transferrin (500m g/ml) 13.5m l of T3 (1m g/ml) 18m l of cholera toxin (50 m g/ml) 5m l of EGF (100 m g/ml)

also;

200m l fungizone (250 m g/ml) 1ml Pen and Strep (100x strength (10000 units/ml and 5000 m g/ml))