Raft culture media¶
Contributed by Matt Lewis <email@example.com>
Raft culture medium, including standard supplements used.
Dilution factors are given here for any quantity but absolute quantities for a target of 100ml raft culture medium are shown in brackets.
- Add 3 parts (70ml) DMEM (including glutamine or glutamax) to a suitable container
- Add 1 part Ham’s F12 (25ml)
- Add 5% FCS (5ml) and mix thoroughly
- Adenine (400ul)
_(Sigma A8626), MW=135, final concentration=180mM, working stock=45mM, Dilution factor=250._
Dissolve stock in 0.05N HCl to 45mM and store working aliquots at –20C.
- Insulin (100ul)
_(Sigma I6634), MW=5733, final concentration=5mg/ml, working stock=5mg/ml, Dilution factor=1000._
Dissolve stock in acidified H2O (0.1ml glacial AcCOOH + 9.9ml H2O) and store working aliquots at 4C.
- Hydrocortisone (40ul)
_(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) and store working aliquots at -20C.
- Transferrin (1ml)
_(Sigma T2036), final concentration=5mg/ml, working stock=500mg/ml, Dilution factor=100._
Dissolve stock in media (DMEM or KGM) and store working aliquots at -20C.
- Penicillin and streptomycin (1ml); and fungizone (200ul)may optionally be added to help prevent accidental infection of cultures.
- T3 (13.5ul)
_(aka 3,3’,5-triiodo-L-thyronine) (Sigma T6397), MW=673, final concentration=0.2nM, working stock=1mM, Dilution factor=7500._
Dissolve stock in 1N NaOH to 1mg/ml. Dilute 1000-fold in 0.1N NaOH and store working aliquots at -20C.
- Cholera toxin (18ul)
_(Sigma), MW=87kD, final concentration=0.1nM, working stock=50mg/ml, Dilution factor=5750._
- EGF (5ul)
_(Sigma E1264), final concentration=5ng/ml, working stock=100mg/ml, Dilution factor=20000._
Dissolve in media (DMEM or KGM) and store working aliquots at -20C.
- Mix thoroughly before use
This method is based, with permission, on an original protocol available here.