Differential staining of RNA and DNA with AO is simple but requires stringent conditions, in particular correct AO concentration in the final staining solution. Dr. KuKuruga correctly emphasizes its advantages and possible problems. The problem with contamination of the instrument is of the same magnitude as using other strongly fluorescing fluorochromes eg. rhodamine 123. I am guessing that the problem of contamination of the instruments by AO was overblown by some companies that sell reagents. The lifetime supply of AO costs about $ 30.- , Considering that AO can substitute for some monoclonal antibodies e.g in discriminating G0 cells, analyzing lymphocyte activation, etc., it is little wonder that the companies selling these antibodies try to discourage the use of AO. We use AO continuously since 1974, in parallel with with many other probes, with different flow cytometers, recently FACScan. A rinse with bleach between the samples, as Dr. KuKuruga points out, is sufficient to remove any contaminating fluorescence. The protocols on use of AO are in Vol 41 of the Methods in Cell Biology (Academic Press, 1994) as well as in Current Protocols in Cytometry. Zbigniew Darzynkiewicz
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