Hi Cheryl, I've seen the same things, and using PI in the same "channel" as another fluorochrome worries me too, although it's possible to mostly compensate it out of FL2 and use FL3 to discriminate live/dead . These intermediates are clearly visible in and annexin V versus PI apoptosis assay and are mostly annexin V high. I advocate the use of 7-AAD along with PE and FITC because it has less of a signal in FL2 even if it's not as bright as PI (Mario). If you've got the luxury of a red laser on your machine then you can use a red-excited dye such as to-pro 3 from molecular probes and have three untainted colours from your 488 laser (as suggested a few years ago on this list by Derek Davies). Cheers, Ray > ----- Original Message ----- > From: "Cheryl Smith" <cheryla.smith@utoronto.ca> > To: "Cytometry Mailing List" <cytometry@flowcyt.cyto.purdue.edu> > Sent: Tuesday, February 13, 2001 1:34 PM > Subject: Viability with PI > > > | > | Hello All, > | > | A couple last questions about PI staining for viability. > | > | I have found that very often there is a population of lower intensity PI > as > | well as the very high one that we typically define as dead. I think there > | are logical explanations for the presence of this, but the problem is how > | can you get away without using a dedicated detector for PI and be sure > that > | some of what you are assuming to be, for instance, positive PE or PE/CY5 > is > | not actually low intensity PI? > | > | I have also seen, even with preparations that have been washed after the > | addition of PI, an increase in the background of the PE signal. I'm not > | really sure why this happens, but do people just ignore it? Wouldn't it > | cause as much problem with samples of not extremely bright, and relatively > | rare, positive populations as would a few, maybe less than 1%, > | non-specifically staining dead cells? And you can't really compensate it > | away. > | > | These things have nagged me for a long time. I hope someone can offer some > | advice. > | > | Thanks, > | Cheryl > | > | > | > > >
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