Hoechst 33258 and 33342 have very different permeability properties; 33342 will get across the intact cytoplasmic membranes of most mammalian cells, while 33258 is typically excluded. If you are working with fixed or permeabilized cells, or with chromosome preps, the two dyes can be used more or less interchangeably; for apoptosis assays, which depend critically on membrane permeability changes, substituting one dye for another is asking for trouble. I also get the impression that if one or two of the published apoptosis assays worked uniformly reliably and well with a broad range of cell types, we wouldn't see new apoptosis assays published every other week. -Howard
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