Raft culture media

Contributed by Matt Lewis <hop2kin@yahoo.co.uk>

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.