Maintenance of CHO cells (RC info Aug 10, 1999)

Medium =

“Alpha MEM plus”

add: 10% HI-FCS

Pen/Strep (5 ml per 500 ml)

Gibco 15140-122 10K units/ml (Pen) and 10,000 ug/ml (Strep)

L-Gln (5 ml per 500 ml)

Gibco 25030-081 (100x i.e. 200 mM or 29.2 mg/ml in 0.85% NaCl)

To split:

Aspirate medium

Wash with 2.5 ml PBS

Add 2.5 ml warm (37 deg C) Trypsin/EDTA and gently rock for 3-5 min (you can see cells lifting up) Gibco 25200-056 (0.25% trypsin, 1 mM EDTA)

Add 10 ml of sterile PBS and pipet up and down to harvest cells.

Spin 1000 x g 5 min

Resuspend in medium (generally use 1/5 vol of original plate, i.e. 2 ml, for new plate)

Frozen Stocks:

· Confluent 15 cm Culture Plate.

· Trypsinize and pellet as usual, then add 10 ml fresh medium containing10% HI-FCS and 5% DMSO (Use fresh/sterile bottle of DMSO that is reserved for tissue culture work.)

· Freeze 0.5 ml aliquots in o-ring tubes at –80 deg for 1-3 days, then transfer to liquid N₂.