Experience with G0/G1 in live cells with flow

Howard Shapiro hms at shapirolab.com
Thu Jan 15 23:00:19 EST 2004


Tim Bushnell wrote:

>I have a user who wishes to look at G0 and G1 populations in live cells.  I
>am wondering if anyone has used the Pyronin Y vs Hoechst in live cells and
>if so, could they share the protocol.	Especially what pitfalls I need to be
>aware of.

In my original procedure, intact cells were incubated with 5-10 µM Hoechst 
33342 for about 5-10 min at 37 °C, after which concentrated Pyronin Y 
(Sigma-Aldrich and Polysciences both produce satisfactorily pure 
preparations of the dye) was added to a concentration of 5 µM and 
incubation continued for 30-45 min. Cells stained with this mixture can 
subsequently be stained with labeled antibodies, provided that the 
concentrations of both Hoechst 33342 and pyronin Y are maintained in all 
staining and washing solutions. When cells are stained with fluorescent 
antibodies and subsequently fixed, Hoechst/pyronin staining is considerably 
easier; dyes are added after the last wash step to achieve concentrations 
of 1 µg/mL Hoechst 33258 or 33342 and 3.3-5 µM (1-1.6 µg/mL) pyronin Y. In 
a dual-beam (UV/488 nm) instrument, the fluorescence of Hoechst 33342, 
excited in the UV and measured in the range between 440-480 nm, is 
indicative of DNA content; pyronin Y fluorescence, measured between 570-600 
nm, is indicative of RNA content. I had no problem staining cells, but they 
didn't survive the procedure.

Edward Srour, of the University of Indiana, who was interested in studying 
the cell cycle in human hematopoietic stem cells, decided to find out 
whether the Hoechst/pyronin DNA/RNA staining procedure would work at lower 
dye concentrations without killing the cells. It did; he and his colleagues 
now routinely sort and culture cells stained with Hoechst 33342 at a 
concentration of 1.6 µM (1 µg/mL) and pyronin Y at a concentration of 3.3 
µM (also 1 µg/mL), adding 50-100 µM verapamil to block efflux of the dyes.

What does this prove? "Hoechst and pyronin don't kill cells; Howard kills 
cells - with Hoechst and pyronin"!

A definitive reference-

Srour EF, Jordan CT: Isolation and Characterization of Primitive 
Hematopoietic Cells Based on Their Position in Cell Cycle. In: Klug CA, 
Jordan CT (eds): Hematopoietic Stem Cell Protocols (Methods in Molecular 
Medicine, Vol. 63), Totowa (NJ), Humana Press, 2001, pp. 93-111

-Howard









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