Re: Dual staining with Hoescht 33342 and PI
Dr Michael Ormerod (mike_o@icr.ac.uk)
Fri, 06 Oct 95 12:06:12 +0100
I have used this method to lookat a murine haemopoetic cell line (BAF3).
I would suggest that you record orange fluorescence (using PE filters) and
look at the blue/orange ratio. The orange signal is brighter and will
give a better balance. The PI positive cells can be reorded on the red
channel (>600 nm) and gated out before you look at the Hoechst spectral
ratio in the cells with an intact membrane.
To detect apoptotic cells, you might consider using less Hoechst 33342
( 1ugml) for a short time at 37C (5-10 min). In some cells, the apoptotic
cells take up the Hoechst dye more rapidly than the normal. I have
published this method in J. Immun. Meth. and have described in more detail
in Cytometry. A similar method using YOPRO-1 and 488 nm light has just
been published by a French group in J. Immun. Meth. I have tried it and
it worked nicely.
Mike Ormerod
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