Re: yeast cell cycle/viability

I've seen this in yeast stained with sytox green ( the fluorescence
beyond the G2 peak) and gated on FL1A vs FL1W.	It may be that the
yeast, instead of dividing after G2 are going through another
replication of DNA.   Jane Miller, Dept. MIcro. Mole. Path., TX A&M
Hlth. Sci. Ctr., College Station, TX  77843

>>> Alicia Bicknell <aware@biomail.ucsd.edu> 1/6/2006 10:15 AM >>>
I am very new to flow cytometry and still trying to figure out some 
basic things.  I am investigating certain aspects of the yeast cell 
cycle.	I am fixing cells in 70% ethanol and staining with styox green.

  Prior to staining, I sonicate at 30% for 15 seconds.	I find that this

amount of sonication is sufficient to disrupt all doublets in a normal

untreated wild type population, so there is no need to gate on singlets

in a FL1A vs FL1W graph.  When I add a certain drug, I detect G2/M 
phase arrest in my cells.  However, I also detect a significant amount

of high fluorescence cells after the drug is added.  If I gate on what

should be singlets in my population, these high fluorescence cells are

eliminated, but I'm not entirely convinced that those high fluorescence

cells are, in fact, cells sticking together, so I don't feel confident

that it is okay to gate them out.  Might these high fluorescent cells 
also be apoptotic cells that are taking in MORE dye because they are 
already permeable prior to fixation?  Any ideas on how to test this? 
The only idea I have is to use a vital DNA dye + the sytox green (in 
this case, I would not fix the cells).	Has anybody had any luck doing 
an experiment like this?
I should note that all of the below graphs contain only "live" cells as

determined by forward and side scatter.

Thanks in advance for any insights,
Alicia Bicknell


Untreated