Propidium iodide will leak out of the "dead" cell population and into the "live" cell population following fixation or else it will simply leak out of all cells if there is not excess PI in the medium. It won't work for this experiment because its affinity for nucleic acids is too low and its off rate too high. However, we have found that ethidium homodimer-2 (Also called Dead Red!) has a relatively slow kinetic off rate and is reasonably slow to transfer from a dead cell to a fixed cell (that was not dead when the experiment started). The discrimination is still clearly apparent after 24 hours, although the differences narrow with time. This also permits Live/Dead discrimination in ALL cell viability experiments on a totally fixed-cell prep, which can reduce biohazards. See this link for the flow cytometric results: http://www.probes.com/handbook/figures/1503.html Spectrally the dye is almost identical to PI and it can be used in combination with any green-fluorescent antibody. We determined that ethidium homodimer-1 is not as good as ethidium homodimer-2 for this assay. http://www.probes.com/servlets/product?region=Select+Region&item=3599 ray hester wrote: > Hi, > > An investigator wants to identify transfected cells with an FITC-conjugated > antibody to an intracellular marker (so the cells will have to be fixed and > permeabilized) and at the same time he wishes to know the percentage of > these transfected cells that are viable/non-viable. > > I assume that propidium iodide is out, since all of the cells will fluoresce > with this dye after fixation and permeablization, however > > I also seem to remember that propidium iodide isn't covalently bound to DNA > and that it will diffuse out over time if the PI-stained cells aren't kept > in PI-buffer, but is it possible to do the transfection, add the PI (I'm not > sure what the time frame is here), fix and permeablize the cells, stain with > the anti intracellular marker antibody, and do a flow analysis before > substantial amounts of the PI have left the dead-as-a-result-of-transfection > cells (the anti intracellular marker antibody doesn't have to be FITC > conjugated, I don't believe, if that helps at all)? > > If the answer to this is, no, are there any dead-cell markers/antigens to > which antibodies are available? > > Thanks. > > Ray Hester > Univ. of South Alabama > rhester@jaguar1.usouthal.edu
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