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

Ive noticed the same thing with a Zinc binding dye on our cyan.  In this 
case the fluorescence steadily drops for the first........10-15 seconds and 
then is perfectly stable.  I'm usually a little skeptical when doing any 
type of analysis where Median or mean is gonna be important so i just 
happened to be looking at a plot ofTime vs fluorescence.

j




At 07:01 AM 12/19/2007, Roberts Joanna wrote:
>
>
>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
>
>
>
>
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James Marvin
Manager, RHLCCC Flow Cytometry Facility
http://www.basic.northwestern.edu/sharedresources/flowcytometry/flowcyometrysite.html
312-503-0913 (office)
312-908-1294 (lab)


"What must a man possess who possesses the Possessor of all things?"
Savanarola