>to whom it may concern >the problem i have is that most people fix yeast cells before adding the >hoechst stain to them for DAPI filter images of the nucleus , but i want to >stain live yeast cells as fixing cells bleaches the GFP fluorescence which i >want to localize to the nuclear region. so i need to get a protocol for hoechst >staining of LIVE yeast cells, without fixing them. i have tried adding hoechst >to a liquid culture but the cells dont seem to take up the dye unless they are >fixed. i would appreciate any suggestions with regard to this problem >thanks >roopa Hallo Roopa, have had a similar problem. I think (could be wrong) most of your dead cells will not be expressing the GFP in the first place: so the dead cells will be the small cells at the lower end of the FSC scale and negative for GFP. Have sorted these and very few grow, whereas there is always good growth for the GFP positives. You could try that, it keeps things simple; unless you need to do cell cycle analysis. There is a Live/Dead Yeast viability kit available from Molecular Probes (based on metabolic activity) but it's not really compatible with GFP staining as it stains the dead cells green and live cells red, and it was difficult to get a good separation between what was double pos. and what was only green on the cytometer. Might be o.k for microscopy. I'm also interested in hearing about a protocol for cell cycle analysis in live yeast, if there is such a thing. Good luck Ann
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