Algorithm for manually setting multicolor compensation rozenkov at
Thu Nov 3 07:08:06 EST 2005

Dear Uriel, 
What you need to do to achieve the matrix algebra compensation manually is just to repeat
compensation circles. Start with FITC and go to the right (red direction) through all
colors ? three or whatever number of channels you have. It is better to move from the
shorter wavelengths (green) to the longer, as fluorescence spectra are usually skewed to
the right by absorbance and there is more spillage to the right. However, when you run
each single stained color you can compensate into all other channels, both to the right
and left.
When you finish your sequence of single color tubes, check the low voltages (unstained or
isotype control tube) if they are still good and then run your compensation tubes again.
When you run a color second time you already have basic compensations from other channels
into this channel, and can see how other channels change compensation needs for this
channel and fine tune it appropriately. Usually, you need to make some slight adjustments
in the second round.
If you want to achieve ideal (or near to) compensations keep repeating the above until
you stop making changes in all channels. This should be hard to do with 17 colors, but if
you have only 3-5 channels it is not too complicated (3-4 circles the most).
Kind regards,
Vladislav Rozenkov, M.D., Ph.D.
Centre for Immune and Targeted Therapy
Division of Medicine
University of Queensland
rozenkov at
-----Original Message-----
From: Uriel TK <utk1 at>
To: cyto-inbox
Sent: Mon, 05 Sep 2005 03:00:14 +0200
Subject: Algorithm for manually setting multicolor compensation

I am doing 3 and 4 color experiments with software compensation and I wanted to ask what
is your "algorithm" for setting compensation manually. Just to make sure you know what
I'm doing this is how I set single compensations: For every color I pick the brightest
expression and use the cells gated negative vs cells gated positive in a dot plot and set
the compensation so that positive cells have the same median (down to the 1st decimal
value) as the negative cells on the compensated channel. I am using FITC in FL1, PE in
FL2 and EMA in FL3 (FACScan or calibur). The question is in what order you set the
compensations, and which compensations do you set? Every color has some spillage into all
the other channels, including FITC into FL3 and EMA into FL1. For example:
1)Do you compensate FL3 for FITC spillage and FL1 for EMA spillage? Or do you just let
the FL2 vs FL1 and FL2 vs FL3 take care of "all"? If you do FL1 vs FL3, do you set it
before or after FL2 vs FL3?
2)Do you do 1st all "forward" compensations (FITC into FL2 (and 3?) and PE into FL3) and
then backward ones (PE into FL1 and EMA into FL2) or do you do all FITC, then all PE then
all EMA?
All these give different comp matrices, but similarly good results (visually and by
medians). These are experiments dealing with population numbers so perfect compensation
is not necessary to be able to separate them, but in the future I will be using
fluorescence values so it is important to set comp accurately. Meanwhile I am not using
automatic compensation, but that is a discussion for another occasion!
Many thanks for your help,
Uriel Trahtemberg
MD/PhD student
The Laboratory for Cellular and Molecular Immunology
The Hebrew University - Hadassah Medical Organization
Jerusalem - ISRAEL 
"God does not care about our mathematical difficulties. He integrates empirically." 
 Albert Einstein
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