RE: yeast cell cycle/viability

Alicia,
Assessment of doublets and apoptotic state is very straightforward with flow
imaging (see George, et.al., Cytometry Part A 59A:237-245, 2004). 
The work cited above was done in Jurkat cells, but we've done lots of yeast
imaging as well.
If you want to run a sample or two on our in-house ImageStream systems to
see what's going on, just let me know.
Best,
David

David Basiji, Ph.D.
CEO
Amnis Corporation
2505 Third Ave., Suite 210
Seattle, WA 98121
(206) 374-7165 direct
(206) 919-3342 mobile
(206) 576-6895 fax
www.amnis.com <http://www.amnis.com/> 

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From: Alicia Bicknell [mailto:aware@biomail.ucsd.edu] 
Sent: Friday, January 06, 2006 8:15 AM
To: cyto-inbox
Subject: yeast cell cycle/viability


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