Kelly, where were you when I was trying to PI stain yeast! That is a million times harder than staining mammalian cells. In general, your protocol looks fine. Perhaps some folks who work with CHO more than I do can help you fine-tune. If you trypsinize, and add the ethanol cold, dropwise, while vortexing, you should be able to eliminate aggregates, as you do by sonicating the yeast. Some people, instead of using EtOH permeabilization, use detergent (NP40) to stip the nuclei out of their mammalian cells. This "Vindelov" protocol can be found in J. Paul Robinson's methods book. You can store the EtOH treated cells in the cold if you wish, but many people have found that they reamian stable for a week or longer (in EtOH) at room temperature, in the dark. Mark
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