Mario Roederer wrote- >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. Mario and Maryalice are both right. Fixation renders cells permeable to PI, but, if cells are exposed to PI and then washed before fixation, cells which were permeable to PI before fixation will retain it for a while, because, while the PI concentration eventually reaches a new equilibrium, this takes a few hours to happen. For what it's worth, the dimeric dyes (ethidium homodimer, YOYO-1, TOTO-1, TOTO-3, etc.) bind much more tighly to nucleic acid than to monomers such as propidium. It is said that a probe labeled with dimeric dye will retain its label even when a huge excess of nucleic acid is added for hybridization. The dimeric dyes should therefore be good labels for "live-dead" discrimination(i.e., for distinguishing cells with and without membrane permeability to those dyes) in samples which are first treated with dye, then washed, then fixed. You can't use the same fluorescence channel as you do with PI, but, if you keep the "dead" cells off scale, that shouldn't matter. >(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). Transient permeabilization, whether of eukaryotic or prokaryotic cells, is a different kettle of fish (or can of worms) entirely. A generation ago, we came up with a dual-staining method to determine the extent of permeabilization and resealing following lysolecithin treatment. Add PI first, treat the cells with lysolecithin, then add fluorescein diacetate (carboxyfluorescein diacetate or calcein-AM would now be preferable due to better retention of the product). Cells which were permeable before lysolecithin treatment (mostly dead cells) will have red nuclei; cells which never became permeable will have green cytoplasm, and cells which became permeable and resealed will have red nuclei and green cytoplasm. However, if you attempt to sort the presumptively viable two-color cells and grow them, you may find that the propidium prevents them from growing... -Howard
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