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> I would like to try a 3 colour experiment. The aim is to study the
> effect of transfected constructs on cell cycle progression. So I need
> to gate for transfected cells so I am cotransfecting a gfp construct
> although I could us a CD2 construct. I need a DNA stain for cell cycle
> analysis and I want to do an Ab labelling as well. Options I have come
> up with so far are 1) gfp(transfected cells); PI (DNA) and Cy5(Ab); or
> 2)Cy3-CD2 (transfected cells) ; FITC (Ab) and Hoechst (DNA)...
Your first problem with option 1 is the difficulty of permeabilizing
the cells for PI staining without destroying GFP. This was commented
on recently on this list by Steven Weintraub (3july), Zbigniew
Darzynkiewicz (6july) and Rochelle Diamond (9july). Apart fom that,
PI will be excited by your second laser (to an extent depending on
whether it is a dye laser or a HeNe) and may swamp the Cy5 signal. We
are assuming you have two excitation time points and that inter-beam
compensation is not possible for you. You may be able to fix this if
you have a second channel at the second time point to independently
detect the PI spillover and then to compensate it out of the Cy5
channel.
Your second option will be ok as long as the Hoechst and FITC
excitations are spatially and/or temporally separated; otherwise, the
Hoechst will spill into the FITC channel to an extent that
fluorescence compensation electronics will be hard pressed to remove.
Also, in principle, this option would allow GFP (transfection), PE or
Cy3 (Ab) and Hoechst (DNA) as an alternative combination.
> Does
> transfection of gfp, or mere transfection alone alter cell cycle
> profiles? Any advice would be appreciated.
*Preliminary* experiments by someone here at WEHI point to GFP
affecting cell cycle determination.
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