I have been staining yeast cells for analysis of cell cycle using SYTOX green and getting good results.
The cells i'm now tiring to stain express GFP so SYTOX green wouldn't work as they emit at the same wave length.
I have tried using both SYTO 60 and DRAQ5 with little success.
SYTO60 seems to mainly stain mitochondria and DRAQ5 does not provide great separation between G1 & G2.
anyone knows about a better protocol for DRAQ5 (for now i've been diluting it 1:1000 in PBS and incubating for 15 min in room temp) or a better DNA stain emitting in far-red and works well with yeast?
Hello
These links will help you
Article Guidelines for the use of flow cytometry and cell sorting in...
Article The Use of DRAQ5 to Monitor Intracellular DNA in Escherichia...
Good Luck
If u are still interested - If I am correct, the cell cycle stain uses some harsh fixation and proteolysis steps which might be incompatible with gfp imaging due to denaturation. Can u present the protocol?
If I wanted to do what you want, I would use an alternative method for cell cycle determination, such as cytometric quantification of histone amounts. So, your protein of interest can be a gfp fusion, while you need to tag a high abundance histone with with an RFP. Ive done cytometry of histone tagged yeast and the n/2n peaks are really nice. I think it was Hta2-GFP
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