I recommend trying our (Molecular Probe's) YO-PRO-1 to detect apoptotic cells in an adherent-cell population (or tissues?). The reagent is a nucleic acid stain that is nonfluorescent except when bound to intracellular nucleic acids. It is selectively permeant to membranes of apoptotic cells and has been used for this purpose several times in flow cytometry and imaging. Since it is also permeant to dead cells, I recommend combining it with propidium iodide to detect any dead cells (which will have bright red fluorescence). Most likely both would be used at about 1 uM or less in standard media (minus phenol red) for perhaps 15 minutes, using a fluorescein long-pass filter to discriminate the staining of apoptotic and dead cells (live cells should be unstained but could likely be stained blue fluorescent with Hoechst 33342). These dyes will probably NOT require any wash step after staining if used dilute enough. Ethidium homodimer-1 at an even lower concentration would definitely not require a wash step to detect the dead-cell population. The flow cytometric version of the assay is described (in the Product Information Sheet) here: http://www.probes.com/servlets/product?region=Select&item=13243 (although I see that the abscissa in the figure in the Product Information Sheet that is now marked "FITC" fluorescence" really would be better marked "YO-PRO-1 fluorescence, although FITC and YO-PRO-1 use the same optical filters and both are FL 1 measurements in a flow cytometry). I would be interested in hearing form anyone who has tried this in imaging of adherent, partially apoptotic cells and we plan to try the 3-color experiment soon. Some Pubmed links on doing this (by microscopy): http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9749586&dopt=Abstract http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7561136&dopt=Abstract http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10870639&dopt=Abstract
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