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

Hi Christopher,

Maybe I'm missing something here, but if one decides to go for your
option 1, what would stop you from washing the cells some more and then
add nucleic acid stain back into the resuspension buffer? These dyes
don't fluoresce significantly without binding nucleic acids, so the
extracellular excess won't contribute to the extracellular fluorescence
you hypothsize about. In contrast, PE/FITC/Alexa dyes/what have you
labels that don't change fluorescence before/after binding would be
washed away, and no longer add noise.

Just my 2c - and Merry Xmas/New Year's to everyone on the list!

Guy

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-----Original Message-----
From: Christopher Bare [mailto:flowmail@verizon.net] 
Sent: Thursday, December 20, 2007 4:32 PM
To: cyto-inbox
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
> 
>