Growth Medium
into 0.5 L Gibco bottle:
500 ml DMEM
25 ml Fetal Bovine Serum (FBS) (5%)
500 ml gentomycin (0.1%)
500 ml penicillin/streptomycin (0.1%)

2x Freeze Medium
24 ml growth medium
8 ml FBS
8 ml DMSO

Trypsinizing Cells and Replating
To release confluent layer of cells on large dish:

• Prepare elastic strips by sterilizing with 70% EtOH and drying thoroughly
• Aspirate out growth medium
• Add 10 ml Ca2+ free medium – 5 minutes in incubator
• Aspirate out Ca2+ free medium
• Add 10 ml more Ca2+ free medium – 5 minutes in incubator
• Aspirate out Ca2+ free medium
• Add 2 ml 0.25% trypsin, distribute evenly by rocking – 2 min. in incubator
• If cells still attached after 2 min, aspirate out trypsin and replace with 2 more ml
• Incubate until cells have rounded up and look like they will release
• Tap dish against microscope to release them from plate
• Once majority of cells have released, add 10 ml growth medium
• Wash plate with pipet several times to knock off all cells
• Transfer to 15 ml tube with pipet
• Spin for 5 min
• Aspirate out liquid above pellet, resuspend in 10 ml growth medium
• Dilute 5 ml of cell suspension into 5 ml growth media in another tube
• Plate 4 ml of diluted cell suspension onto small dishes with sterile strips already in place
• Cells should be close to confluent in 24 hours.
• *[FBS] can be bumped up to 10% if faster cell growth is desired

Thawing Frozen MDCK Cells

• Get cells out of N2 (l) canister
• Thaw cryovial in 37 degree bath
• Add contents of vial (about 1 ml) to petri dish
• Add 20 ml of growth medium
• Change medium as soon as cells have attached (several hours)