> We are interested in using Pyronin Y (PY) staining of RNA to distinguish G0 >phase from G1 phase hematopoietic cells. A recent publication suggested that >the concentration of PY had to be <5 micrograms/mL to maintain viability but >that at concentrations of about 1 ug/ml, PY preferentially stained >mitochondria. Does anyone have experience with this stain? Can they >recommend a reliable PY concentration for staining RNA "specifically" ? What >incubation period and temperature are used for PY entry? Does PY have any >effect on cell physiology at "nontoxic" concentrations? Thanks for the >advice!! Pyronin Y is, as far as I know, pretty toxic to human cells. When I developed the Hoechst 33342/pyronin Y stain for DNA and RNA in unfixed cells in 1980, I thought it might be usable for vital sorting on the basis of DNA and RNA content, enabling isolation of cells in the subcompartments (G0/G1A/G1B) of the cell cycle previously defined by Darzynkiewicz and colleagues using acridine orange; the AO staining protocol required killing the cells. It was established that Hoechst 33342 at the 3-10 uM concentrations used wouldn't kill cells, and that they could retain viability after sorting provided low power UV excitation (arc lamp or <50 mW from a UV laser) was used. We could wash Hoechst 33342-stained cells and culture them; however, cells stained with the Hoechst dye and 5 uM pyronin or with 5 uM pyronin alone were killed. Darzynkiewicz et al later established (although I don't recall offhand in which of their works on the subject it was published) that pyronin Y at 5 uM killed cells by complexing irreversibly with double-stranded RNA. Pyronin will enter living cells with intact membranes; it is pumped out by the MDR efflux pump, as are Hoechst dyes. This pump does not seem to be terribly active in resting human T cells; it is more active in B's, activated T's, and, of course, in the CD34+ stem cell population also noted for low intensity of Hoechst 33342 and rhodamine 123 staining. The RNA specificity of pyronin Y staining in intact cells is essentially impossible to establish because one cannot readily ribonuclease treat those cells; ethanol fixation and RNAse treatment were used to show that at least 80% of pyronin fluorescence in cells was eliminated by RNAse treatment. Resting T cells do not have very active mitochondria (i.e., they don't stain brightly with rhodamine 123), and pyronin Y fluorescence in live resting cells is predominantly from the small amount of RNA present. When cells become activated, the mitochondria become more active, and RNA is synthesized; in principle, increased pyronin Y fluorescence in intact activated T-cells could be explained by either or both of these effects. However, in my experience, poisoning the mitochondria with metabolic inhibitors doesn't significantly decrease pyronin Y fluorescence when 5 uM dye is used, suggesting most of the staining is of RNA. I used 30 min incubation periods at 37 C with 5 uM dye for staining viable cells. It is necessary to use either Hoechst 33342 or methyl green to block DNA staining by pyronin Y, whether cells are live or fixed. In fixed cells, good DNA/RNA staining is obtained after a few minutes' incubation with 500 nm pyronin Y and 1 ug/ml Hoechst 33342 at room temperature. -Howard
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