FACSDiva autocomp

Dear Flow Experts,

 

I'm wondering if I'm setting up the autocomp incorrectly for one of my
customers.  I've been reading about biexponential transformation,
autofluorescence and fluorescence minus one controls in the list
archives.  We're using a FACSAria with FACSDiva 4.0.  I'll be getting
the latest version of the software (with biexponential capability) soon.
I'm not sure if I'm setting the negative wrong, or if it's something
else.  I was under the impression that I should set the negatives in the
first decade or so, no matter what, and go from there.  Based on what
I'm reading now, though, that isn't necessarily right.  

 

The experiment is staining murine iliac node cells with various
combinations of FITC, PE, APC, PE-Cy5 and DAPI.  After the autocomp is
set and I re-run the single-stained samples with compensation, the PE is
almost always over-compensated in the FITC channel.  I use the
fluorescent means of the negative and PE-positive populations in the
FITC channel to adjust the compensation after autocomp.  The FITC is
also often under-compensated in the PE channel.  I've also noticed some
problems with PE vs. PE-Cy5.  

 

I'm seeing similar problems (FITC vs. PE) with the compensation of
murine genital tract cells stained with FITC, PE, APC and DAPI.  (We're
focusing mostly on lymphocytes from the nodes and the genital tracts.)  

 

My questions are:

 

Is it ok for me to fiddle with the compensation to get the FITC vs. PE
and other combinations to look right using the mean fluorescent values
after autocomp?  

 

Any ideas about what I might be doing wrong?

 

If we try the FMO controls, how does that work?  As I understand it, the
FMO is used to set the "true" negative for the missing
fluorochrome-right?  So, do we use the FMO first to get the true
negative for each channel, and then do the single-stained controls using
the autocomp?  Each fluorochrome is used with more than one antibody-can
we pick one for each FMO or do we need to evaluate each antibody?   

 

Thanks in advance for your help!

 

Happy Holidays!

Robin

 

Robin Stingley, Ph.D.

Flow Cytometry Core Facility

Department of Microbiology and Immunology

University of Arkansas for Medical Sciences

Slot 511, Rm B504

4301 West Markham Street

Little Rock, Arkansas 72205

Phone:	501-686-6927

E-mail:  StingleyRobinL@uams.edu

http://www.uams.edu/flowcytometry/ <http://www.uams.edu/flowcytometry/> 

 

 


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