WEHICM

4/5/94

MAKING WEHI CONDITIONED MEDIA FOR

GROWTH OF 32-D OR FDCP CELL LINES

A. The cells grow in RPMI 1640 supplemented with 10% FBS and Pen/Strep solution: Use Sigma hybri-max P/S solution and add 1% to GIBCO media (which is unsupplemented) or 0.5% to Joslin TCC media.

Thaw the cells in 37 C water bath (no shaking). If only a few cells survive its OK: they will come back. They grow fast. Once at sufficient density, they double every 14-15 hours (estimate). They will die at densities >= 2 x 10⁶/ml.

They are suspension cells. Grow them in flasks and split by resuspending settled and quasi-attached cells and transferring some to a new flask with new media.

B. To make conditioned media, grow to maximal confluence and when cells begin to die harvest media. Spin out dead/dying cells and filter 0.2 m. WEHI conditioned media contains lots of IL-3. Stable at 4 C for several months. Protect from light.

C. Freeze log-growth cells by spinning down 10-20 mls of cells and freezing 3 x 1.5 ml cryovials in RPMI + 20% FBS + 8% DMSO. Freeze -20 C 1 hour, -70 C overnight and transfer to liquid nitrogen..