Griffin:Conditioned Medium Preparation

Protocol

• Culture cells in appropriate size flask (T75, T125, or larger) under normal culture media (DMEM & FCS + penn/strep) until optimal confluency (The confluency for cells at changing to serum free media varies with each cell line, where preserving the phenotype while minimal cytoxicity or cell death is relevant; ~80-100%).

• Aspirate off the normal growth medium (FCS/penn/strep), wash monolayer 3x sterile PBS, and replenish cells with serum-free (SF) media.

Ideally cells will be 100% confluent with out too many dead or lysed cells at the time of harvesting. After ~48hours incubation in SF media, cells and the conditioned media (CM) are collected as follows.

Sterile-filter conditioned media by pouring it from the flask directly into a bottle top 0.22 micron filter while under vacuum, filter into a sterile bottle. Dispense 30 ml aliquots into sterile 50 ml tubes. Dispose of flasks containing cells.

Before collection of the conditioned media (CM),