Algorithm for manually setting multicolor compensation

Gerstein, Rachel Rachel.Gerstein at
Fri Nov 4 10:09:29 EST 2005

the alternative for all this (for analysis samples) is to collect uncompensated data and
then use FlowJo for software compensation

Rachel M. Gerstein, Ph.D.
Assistant Professor
Department of Molecular Genetics and Microbiology
Graduate Program in Immunology/Virology
University of Massachusetts Medical School
55 Lake Avenue North
Worcester, MA 01655-0002
(508) 856-1044
(508) 856-5920 (FAX)

> ----------
> From: 	rozenkov at
> Sent: 	Thursday, November 3, 2005 7:08 AM
> To:	Cytometry Mailing List
> Subject:	Re: Algorithm for manually setting multicolor compensation
> 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 
> Vladislav Rozenkov, M.D., Ph.D.
> Centre for Immune and Targeted Therapy
> Division of Medicine
> University of Queensland
> Brisbane
> Australia
> rozenkov at
> -----Original Message-----
> From: Uriel TK <utk1 at>
> To: Cytometry Mailing List <cytometry at>
> Sent: Mon, 05 Sep 2005 03:00:14 +0200
> Subject: Algorithm for manually setting multicolor compensation
> Hello!
> 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.
> 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
>   _____  
> Look What The New Can Do!
> Now you can preview dozens of stories and have the ones you select delivered to you
without ever leaving the Top Home Page. And the new Tool Box gives you one click access
to local Movie times, Maps, White Pages and more. Click to test drive. 

More information about the Cytometry mailing list