RE: 7AAD and PY for G0 and G1 - signal fluctuation at beginning of analysis

Joanna:
Joanna:
We too have seen what you are talking about on the Cyan and like Chris we
have also seen the same phenomena with certain fluorochrome conjugated
antibodies which when washed goes away. I think Chris' assessment is
absolutely correct as we were able to diminish it by changing the core size
or washing the sample. Interestingly we have not experienced this with PI or
DAPI but have with 7-AAD and TOPRO-3 (see attached Word document). You
should be able to gate the stable fluorescence on a plot of intensity vs.
time or wait for it to stabilize. For fluorochrome conjugates washing does
the trick but as Chris mentions that is probably not the best approach for
nucleic acid dyes. 
I brought this up to DAKO several years ago and they didn't seem to have an
answer and claimed that I seemed to be the only one seeing such a problem.
FWIW-Joanne


Joanne Lannigan, MS
Director, Flow Cytometry Core Facility
University of Virginia
Jordan Hall, Room 7067
P.O. Box 800734
Charlottesville, VA 22908-0734
Office: 434-924-0274
Lab: 434-243-2695
Fax: 434-982-1071
email: joannelannigan@virginia.edu

> -----Original Message-----
> From: Christopher Bare [mailto:flowmail@verizon.net]
> Sent: Thursday, December 20, 2007 10:32 AM
> To: Cytometry Mailing List
> Subject: RE: 7AAD and PY for G0 and G1 - signal fluctuation at beginning
> of analysis
> 
> Joanna,
> 
> I have seen a very similar effect when running samples containing a large
> excess of streptavidin-PE on an Influx.
> 
> To add to the observation, I found the final level to be core differential
> dependant: higher core pressure increase my final stable relative
> fluorescence level concomitantly.
> 
> Here's my theory:
> 
> This is related to excess fluorescent molecule in the core. The excess
> fluor
> is surrounding the cells and contributes to the area measurement but is
> not
> actually bound to the cell.
> 
> Hence, the effect is not seen with beads since there is no extra-particle
> fluorescence. It is also not as evident when samples are washed prior to
> acquisition (my observation), but more noticeable with high excess of
> fluor
> (such as with nucleic acid stains or my own overkill SA-PE).
> 
> Solutions would be:
> 
> 1. wash samples before acquisition to remove excess fluor -- but this can
> diminish nucleic acid stains that need to be in equilibrium.
> 
> 2. wait until event rate and RFI have stabilized before acquiring or
> sorting
> -- but this is difficult with small samples
> 
> 3. run at a smaller differential (slower rate) to minimize core diameter -
> -
> but this adds precious time to experiments.
> 
> Anyone else have suggestions?
> 
> -Christopher Bare
> Rockefeller University
> Flow Cytometry Resource Center
> 
> > -----Original Message-----
> > From: Roberts Joanna [mailto:joanna.roberts@epfl.ch]
> > Sent: Wednesday, December 19, 2007 8:02 AM
> > To: Cytometry Mailing List
> > Subject: 7AAD and PY for G0 and G1 - signal fluctuation at beginning of
> analysis
> >
> >
> >
> > Dear Flow people,
> >
> >
> >
> > As always, i am so glad that I can direct my questions to a bunch of
> > people with lots of good ideas.
> >
> >
> >
> > I have a customer doing an experiment on a cell line looking at cell
> > cycle and G0/G1 using 7AAD and PY. The experiment is being run on a
> > CyAn. The problem we are seeing is that for the first few seconds of
> > data acquisition, the signals start low and increase to a stable level.
> > This seems unexpected. Has anyone seen anything like this? The early
> > parts of each measurement have to be discarded. It seems that these
> > signals should be stable throughout and not fluctuate.
> >
> >
> >
> > Here are some other details of experiment set up
> >
> > -	   using low flow rate to get good CVs (at this flow rate, there
> > is a delay of about 30 secs between placing cells on the machine and
> > seeing data)
> >
> > -	   measuring linear area signals
> >
> > -	   triggering on 7AAD channel (but if we use FS we still see the
> > same thing)
> >
> >
> >
> > What seems really weird to me is that
> >
> > -	   running the same sample over and over again gives the same
> > result each time (low signals quickly climbing to a stable level) SEE
> > TOP LINE OF GRAPHS IN ATTACHMENT
> >
> > -	   this effect is not at all noticeable when running alignment
> > beads BOTTOM LINE OF GRAPHS IN ATTACHMENT
> >
> > -	   if you stop acquisition, leave tube on machine, scratch your
> > head, and then start acquisition again, the signals are STABLE (no
> > fluctuation)
> >
> > -	   effect is most obvious in PE channel (for PY) but is also
> > visible in the PECy5 channel (7AAD)
> >
> >
> >
> > Because I don't see this effect with the alignment beads, I find it hard
> > to believe it is the machine but then I can't think of an explanation in
> > biological or physical terms either. Can anyone help?
> >
> >
> >
> > I will be delighted to hear any thoughts about this.
> >
> >
> >
> > Best wishes,
> >
> > Joanna
> >
> >
> >
> >
> >
> > Flow Cytometry Core Facility
> >
> > EPFL, Swiss Federal Institute of Technology
> >
> > SV-SG, Station 15
> >
> > CH 1015, Lausanne
> >
> > Switzerland
> >
> >
> >
> > +41 21 69 39 547
> >
> >


This attachment - 'Topro.doc' -  50.18 KBytes - can be viewed at
http://www.cyto.purdue.edu/MD-parts/530489bc6cb31395b144d6653062623a1ab23bf7.doc