> 1. When viable cells are labeled for RNA content with pyronin Y and sorted >and returned to culture, how long does the stain remain associated with the >cells? I ask because of some concern that pyronin fluorescence might >interfere with subsequent antibody staining and analysis. Although I developed the Hoechst-Pyronin method in hopes of being able to sort viable cells based on DNA and RNA content, I found that the concentration of pyronin I needed to stain viable cells (5 uM) killed the cells. This question pops up on the Mailing List every now and then, and I don't recall anybody ever saying they managed to keep pyronin Y-stained cells reproductively viable. I'd certainly love to hear about it if somebody has had a more positive experience than mine in this regard. > 2. Is it possible to read pyronin Y staining after fixing with 0.5-1.0% >paraformaldehyde? Yes. You don't need to stain until after fixation. I usually add 1 ug/ml Hoechst 33342 and 500 nM pyronin Y to fixed cells, and Triton X-100 to a final concentration of 0.1% to improve permeability. The Hoechst dye (or another DNA dye, such as methyl green) is needed to block pyronin staining of DNA. However, Hoechst dye appears to combine with some reaction product of the fixative, producing strong background fluorescence in the FITC and pyronin (or PE) channels. Pyronin also produces crosstalk in the FITC and PE-Cy5/PerCP channels, but dropping the concentration below 500 nM results in unacceptably low RNA specificity. -Howard
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