PREPARATION OF CHONDROCYTE CELL

CULTURE MEDIUM
Thaw the FBS and Pen/Strep when they arrive, and aliquot them
(separate them into individual tubes containing 50ml (50 ml Falcon tubes) of
the FBS and other tubes containing 5ml of the Pen Strep (7 ml bijou tubes).
Make it up in the bottle that DMEM comes in
The composition of the culture medium is:

450 ml Dulbecco's modified Eagle's medium

50 ml Fetal Bovine Serum (FBS - F7524)

5 ml of Penicillin-Streptomycin (10,000 IU /ml Penicillin and
10 mg/ml Streptomycin)

After making up the medium in the DMEM bottle, pass it through the Falcon
Bottle-Top filters and into 50ml Falcon tubes. now it is sterile!
Store it in the 50ml falcon tubes if not used immediately. The way you know
if it has gone bad is if the medium changes colour- it should stay the same
pink.

PREPARATION OF OSTEOBLAST
CULTURE MEDIUM
On their delivery, store McCoys 5A medium in the fridge (4 C), FBS and Pen-Strep
aliquots in the freezer, and the Vitamin C powder bottle on the shelf at room temperature.
Thaw the FBS and Pen-Strep prior to making up the medium (FBS takes ~4 hours to
thaw on the bench at room temperature).
The composition of the Osteoblast culture medium is as follows :

500 ml McCoys 5A medium

50 ml Heat Inactivated Fetal Bovine Serum (FBS)
0.5 ml of Penicillin-Streptomycin (10,000 IU /ml Penicillin and 10 mg/ml
Streptomycin) (this is the same antibiotic as used in the chondrocyte medium, this may be
changed at a later date)

30 g/ml Vitamin C (L-Ascorbic Acid Phosphate Magnesium Salt nHydrate) (so 16.5 mg is required for a 550 ml portion)

Make the medium up in the McCoys 5A medium bottle. Weigh out the vitamin C into a
6ml Bijou tube, and rinse it out with McCoys 5A from to ensure that all the powder is
transferred into the mixture.
Use a 10 ml pipette to add the FBS, and a 1 ml pipette tip for the Pen-Strep. Once all of
the components have been added, put the lid on the bottle and shake it gently to ensure
complete mixing and that the Vitamin C powder has dissolved.
After the medium is made up, before use it must be passed through 0.22 m filters to
ensure that it is sterile.
Store the medium in labeled 50ml Falcon tubes in the yellow racks in the fridge (at 4 C)
if it is not for immediate use. The medium has a straw yellow/orange colour, though
begins to turn red on contact with air.

Ordering Information:
McCoys 5a Medium - (Invitrogen: 26600-023)
FBS (heat inactivated) (Invitrogen: 10108-157)
Vitamin C (Wako GmbH: 013-19641)