facs help

Mark Peggie mwpeggie at bad.dundee.ac.uk
Wed Aug 16 03:15:42 EST 2000


I routinely spin down 1ml of 5x106 cells and resuspend the pellet in 1ml 70% ethanol, vortex for 10 seconds 
and store at -20 ( Ive used cells after 5 months and they give a resonable FACS profile.  I use 0.5ml of 
these cells, spin them down gently and wash in 1ml 50mM Na Citrate.  Resuspend the pellet in 0.5ml of 50mM Na 
Citrate containing 0.1mgml RNase and incubate at 37oc for 2 hours.  I then sonicate the samples for 30 secs 
to break apart any cells that may have clumped and add 0.5ml 12ugml PI.  I have found that cells arressted in 
HU for extended periods give increasing sized extra peaks less than 1N.

good luck!

Mark







To:            yeast at net.bio.net
Date:          15 Aug 2000 00:58:12 +0100
From:          Gina Alvino <alvino at u.washington.edu>
Subject:       facs help


Hello Yeast People!

I am working on a strain of S.cerevisiae with a deletion in a single
gene.  This deletion causes the cells to become enlarged with a slower
cell cycle as compared to the isogenic wildtype.  Facs analysis on these
cells is difficult to interpret because the peaks are broader and the
profiles are just plain ugly.  When the cells are challenged with
temperature or drug, the profiles are simply a mess.  I have tried lots of
ways to adjust the profiles when scanning but nothing helps.  So I'm
curious to try different protocols for preparing the cells for
scanning.  My protocol calls for fixation in EtOH at 4C overnight.  The
cell pellet is treated with RNase at 0.25mg/ml at 50C for 1hr.  Proteinase K
is added and incubation goes for another hour at 50C.  The DNA is then
stained with propidium iodide and the cells are stored at 4C until ready
to use.  I have scanned on the same day or days later with no change in
the profile.

Suggestions?

Any thoughts and/or comments would be appreciated.

Gina Alvino
University of Washington
Seattle, WA  USA



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Dr Mark Peggie
Department of Biochemistry 
University of Dundee
MSI/WTB Complex
Dow Street
Dundee
DD1 5EH

Tel/Fax: 01382 344782


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