cell cycle analysis with PI

Albert D Donnenberg, PhD donnal at novell1.dept-med.pitt.edu
Fri Nov 22 16:46:23 EST 1996


Flow'ers

Thanks so much for your advice on how to sharpen PI cvs when staining 
for DNA content and intracellular proteins.  Here is a compendium of 
the responses:

From: R.Knuechel, MD
Institute of Pathology
University of Regensburg
<ruth.knuechel-clarke at klinik.uni-regensburg.de>
First of all PI staining quality is dependent on 
cellular fixation, as you probably know, and fixation in methanol
(cold 70% ) gives the nicest results for a lot of people including us.
When nuclear antibodies are included, we have good experience with a
paraformaldehyde/Tween protocol For your nuclear antibodies, you
probably stain after you have prepared nuclear suspensions? The
quality of this also effects the CV, and also, although it may be
trivial the cell number you have availabel for your analysis.

From: jim phillips
univ. of miami
jphillip at mednet.med.miami.edu
Check your fix. ethanol at 70% in water for a min. of 18 hrs. Put the
pi into the sample only 20 min to an hour before you run the sample. 
Make sure the tip and the machine are clean, run some 20% bleach
through the system to clean the nozzle and get rid of protein.  run
your sample on low or at a slow rate.  do some live gating on width
and area plots.  this gets rid of the doublets.  I hope that this
helps some.  


From: Lisa Adams
LAADAMS at am.pnu.com
It may be dependent on cell type and/or your fixation protocol.  I
have found there is a trade-off between good antibody binding and good
DNA histograms when I use paraformaldehyde as a fixative (which is
what works best in my hands).  It has been my experience that when I
start doing 2- or 3-color analysis, I can't have my cake and eat it
too.

From: "Martin, Jill V." <martin.jill at mayo.edu> (Jill Martin)
I am sure that you have checked for doublets, and that you have a low
flow rate, but these would be two sources of CV error.

From: tom_frey at bdis.com
You don't mention whether you are using an ethanol technique or one of
the now-more-common PFA/detergent methods or products.  Length of PFA
fixation and length of time in PFA free buffer after staining can both
alter PI CVs.  See Cytometry 12:279 for example.
*******************************
Albert D. Donnenberg, PhD
Associate Professor of Medicine
University of Pittsburgh
W1056 BST
200 Lothrop St.
Pittsburgh, PA 15213
(412) 624-9596
*******************************



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