Fw: 7-AAD COMPENSATION PROBLEM

Since no one else seems to be offering a decisive answer on this, I'd like
to take a stab at it, just to see if my more learned brethren flowlosophers
can confirm my thoughts.  It seems that the crux of the matter is: "how do
you adjust compensation on a fluorochrome that is usually read on the linear
scale (e.g. PI, 7-AAD, pyronin Y)?" The tutorials that we often get directed
to describe the process for FITC and PE -- fluorochromes that are read on
log scale where unstained and stained cells fit on the same histogram. So
now what do you do when you're reading on a linear scale, as you would be
for cell cycle analysis, and your fluorochrome (eg 7-AAD) spills into
another channel? Compensation is dependent on the PMT voltages and you have
to change them in order to get unstained cells off the left axis and visible
on the histogram.
 Solution: Set the amps for linear and find the voltages that put your
stained cells in the middle of the histogram where you want them. Now change
to log scale, run your unstained cells and setup quadrants. Your unstained
cells probable won't be all the way into the lower left where you normally
put them but that's tuff, you can't change the PMT voltages! Run your
unstained cells and make a note of the X and Y means (or medians)  and with
the amps still on log, put on your singly stained compensation controls and
set the compensation values so that the stained cells have the right X or Y
mean value as you would normally set compensation. Once set, switch back to
linear scale and your PMT voltages and compensation settings should be
appropriate......  RIGHT???


Dan Rosson PhD
Flow Cytometry Facility
Kimmel Cancer Center
Thomas Jefferson University




----- Original Message ----- 
From: "Lydene McArthur" <lydene.mcarthur@cdhb.govt.nz>
To: cyto-inbox
Sent: Sunday, January 30, 2005 3:37 PM
Subject: Re: 7-AAD COMPENSATION PROBLEM
>
> >>> "Kotsianidis, Ioannis" <i.kotsianidis@imperial.ac.uk> 01/27/05
> 04:29a.m. >>>
> Dear Flowers,
>
>
> I am trying to perform 7-AAD staining for cell cycle with simultaneous
> surface staining.
>
> When I tried to compensate 7-AAD (linear, FL3) with PE (log, FL2),
>
>
>  1) I wasn't able to see any event on an FL2 vs FL3 plot, while
> running
> the PE sample and
>
>
>  2) When I removed the FL3 signal out of FL2 while running the 7-AAD
> tube, I lost all the PE positivity,
>
> both in FL1 vs FL2 plot and my final sample (stained with FITC,PE and
> 7-AAD).
>
>
> I also observed the same phenomenon with APC when I tried to perform
> 3-color analysis with 7-AAD.
>
>
> Can someone please tell me what am I doing wrong? I've read the papers
> by Rabinovitch (1986) and also Schmid (1991)
>
> but they don't seem to worry at all about compensation problems...
>
>
> Thanks in advance,
>
>
> Dr Ioannis Kotsianidis
> Clinical Research Fellow
> Department of Haematology
> Faculty of Medicine - ICSTM
> Hammersmith Hospital
> Du Cane Road
> LONDON W12 0NN
> U.K.
>
>
>



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