Figure 5.

Analysis of the modes of cell death following freezing using a single or repeat freeze protocol. The MCF-7 cells were exposed to a single or repeat freeze to −10°C or −15°C followed by passive thawing and then assessed for apoptotic, necrotic, and living populations using YoPro-1, Propidium Iodide, and Hoechst 33258 (green, red, blue, respectively) at 1, 4, 8, and 24 hours post-thaw. Image analysis of −10°C samples revealed a significant increase in necrosis and a low level of apoptosis during the post-thaw interval following both a single and double freeze. Increased levels were noted in the double samples resulting in increased cell death. Analysis of −15°C samples revealed a marked increase of apoptosis and necrosis compared with −10°C samples, most notably at 4 and 8 hours post-thaw. Repeat −15°C freeze samples revealed increased cell loss and necrosis characterized by reduced apoptosis over the entire time course.