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

  



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