FMO - compensation

Bushnell, Timothy Timothy_Bushnell at URMC.Rochester.edu
Fri Dec 3 17:12:34 EST 2004


Hello Kim:

I do a lot of rare event identification and sorting, and use single stained
controls for compensation, and FMO's as a gating control.

The purpose of an FMO control is to examine and properly set the gates for
positive and negative expression.  

The purpose of the single stained controls is to properly set the spectral
overlap of a given flurochrome in other detectors.  

Therefore, you are correct in the fact that you need single color
compensation controls to establish the compensation settings for your
experiment.  The FMO controls are necessary for properly setting the gating
of positive vs negative populations.  

If you go to the WNYFUG website,
http://www.urmc.rochester.edu/wnyfug/wilshire-2004.pdf

there is a presentation by Jennifer Wilshire on compensation and it
discusses both single color controls and FMO controls.	

I hope that this helps.  If you have more questions, please don't hesitate
to contact me.	

Tim

Timothy Bushnell, Ph.D.
Director, CHGMPD Flow Lab
University of Rochester
tel: 585-273-1361
http://www.urmc.rochester.edu/wnyfug/
 

-----Original Message-----
From: Lowe, Kimberley [mailto:Kimberley_Lowe at dfci.harvard.edu] 
Sent: Thursday, December 02, 2004 3:50 PM
To: cyto-inbox
Subject: FMO - compensation

Hi there,
Just a quick question.	Our group has been doing 'compensation' with FMOs
(Flourescence Minus One).  I have been saying that we really need single
positive controls to do the compensation accurately, but, because we are
looking
for very rare cells, my boss has argued that they want to take into account
all
the color interactions when compensating.  It is very difficult (for me) to
do
compensation this way when I have been use to using single colors. What do
others think?  We are looking for a population that is .01 % of the entire
population and we are using 4 colors - Fitc, PE, PerCP and APC on a two
laser
Calibur.
Thanks so much for your responses!
Cheers,
Kim Lowe
Childrens Hospital
Boston, MA




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