Mario, What cells did you test this on? This goes against my observations but I know you are thorough.We always use it with live cells. Maryalice >I hesitate to disagree with Maryalice, especially given her powerful >admonition that whoever says otherwise knows absolutely nothing >about PI exclusion... but... "he who hesitates is last". > >Actually, we found that we can stain with PI, then fix with 0.5% >paraformaldehyde, and have the ability to discriminate live/dead for >about 2 hours afterward (perhaps as long as 4-6 hours). Waiting >overnight, however, is right out--the PI leaks out of dead cells >(and, if present in the medium, leaks into live cells). As Mark >points out, you should add the PI before the PF, and if you need to >wait more than several hours, use EMA (which is considerably less >practical for various reasons). > >We tested this extensively, because of the importance of doing >live/dead discrimination, as well as the practicality of fixing >cells (for example, from infectious samples). > >Note that we did not test higher concentrations of paraformaldehyde >or other fixatives. > >mr > >(PS, with regard to removal of adherent endothelial or tumor cells >becoming PI+ : note that a variety of protocols, as asserted already >on this list, can transiently permeabilize cells. I would try >removing the cells, washing them well in regular medium, waiting 30 >minutes, and then adding PI). 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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