Time-dependent increase in cell death, cleavage of caspase and visual assessment of cell death. Cell viability with ED77 cells over time with differing oxygen concentrations was measured at 1% O2 (B), 5% O2 (C), and 21% O2 (A, D). (A) Representative raw data plot for 21% O2 incubation at different durations. Cells were incubated for the times stated under the oxygen tensions indicated in the figure. Hatched shaded bars represent cells with intact ψm (live cells); darkly shaded bars represent cells with dissipated ψm (dead cells). Minimum n = 3 Error bars are ± SEM. (B) *p < 0.05 versus 12 hr (intact); +p < 0.05 12 hr (dissipated). (C) Difference in all test groups did not reach statistical significance. (D) Difference in all test groups did not reach statistical significance. (E) First trimester trophoblast exposed to 1%, 5%, and 21% for the time periods shown. Cell lysates were prepared in RIPA and equal amounts (50 µg) of protein were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies to the cleaved active form of caspase-3 and caspase-9. Similar results were obtained in two separate experiments. (F) Cells were loaded with DIOC6, exposed to 21% O2 for the time specified in the figure then stained with propidium iodide and were placed on chamber slides and visualized using Nikon fluorescence microscope. (I) 12 hr; (II) 24 hr; (III) 48 hr. Original magnifications ×100. (IV) Individual live trophoblast. (V) Trophoblast undergoing apoptosis. Original magnifications ×400.