Fig. 3. Induction of apoptosis by conditioned medium. Cultures were grown under hypoxia with or without medium change. After 48 h, the medium was removed, and the cells were analyzed for DNA fragmentation (top panel). The conditioned media was centrifuged at 800 g for 5 min to pellet cells and debris, added directly to a second set of plates, and these were incubated in air or under hypoxia as indicated. After 24 h, these cells were also harvested and analyzed for DNA fragmentation. [pH]o was measured in all cases immediately before harvesting the cells. Note that the control samples shown in the last two lanes of the bottom left panel did not receive conditioned medium. Results are representative of three separate experiments.