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