METHODBOOK

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=180 mM, 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=1 mM, 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))