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).
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