I think Howard, as usual, is right. The conditions and cells studied are important in what you get with PI staining post paraformaldehyde fixation. We have found that peripheral blood cells stain with PI post paraformaldehyde fixation. We have used 1% and 0.5% paraformaldehyde. We even tried to use this many years ago. We took peripheral blood cells, stained with antibodies, lysed and fixed. Then after running them (at least 1 hour and almost always more than 2 hours after fixation), we would add PI and let the cells sit. The object was to see if we could determine the immunophenotype of an unknown tumor and then add PI to the tube with the appropriate markers and get an idea about cell cycle in the tumor cells. You can get an idea about cell cycle that way, how ever it was just a faint idea and I'm too compulsive to find that adequate. I do know that under these conditions we get PI staining of tons of cells, basically all the lymphocytes. Maybe some of the variability we saw was due to fixation for less than 2 hours, although we rarely ran the cells at that point, since longer fixation is better. Other cells may behave differently- if it isn't hematopoietic I'm not really interested. Maryalice Maryalice Stetler-Stevenson Director Flow Cytometry Unit Laboratory of Pathology, NCI, NIH "Learn the rules so you know how to break them properly." The Dalai Lama
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