RE: macrophage autofluorescence

Hello Vladislav,


Thank you for the reference.  As you have suggested, I am providing
additional information regarding my set-up.  Unfortunately I do not have any
images I can provide.  I am running my samples on the FACSAria.  With
macrophages kept in culture over 4 weeks I have significant autofluorescence
with all colors - even the red and violet laser.  I do not have this issue
with cells in culture less time or freshly isolated cells.  These cells are
also infected.	I am using FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, Cy5.5,
APC-Cy7 and Pac Blue.  All are cell surface antibodies with the exception of
Annexin V which I have on Cy5.5.  What I was taught was to bring the PMTs
down for each color while running unstained cells; I then run my
compensation tubes which are usually prepared with the BD Comp Beads.  Due
to the high autofluorescence of my unstained cells the voltages are brought
down so low as to bring my positive peak from my comps into the negative
area.  The cells are washed before and after antibody incubation with FACS
wash prepared with phenol red-free HBSS and fixed with 1% PFA.


Thanks to everyone for their helpful suggestions.  

Tracy


  _____  

From: rozenkov@netscape.net [mailto:rozenkov@netscape.net] 
Sent: Tuesday, June 27, 2006 7:53 AM
To: cyto-inbox
Subject: Re: macrophage autofluorescence


Hello Tracy,


I have recently come across an exploration "Autofluorescence: Causes and
Cures" that you can find at:


http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf


In general, it is not easy to deal with autofluorescence, but I think you
may get a better applicable advice for your cells if you show your picture
and tell what fluorochromes and antibodies you are going to use. Also how
you process the cells, fix, etc? Another question is what flow cytometer you
are using, i.e. what channels you have available.


You may be able to "shift" the positively stained cells from the
autofluorescence tail by combining the antibodies of unequal antigen
expression intensity or use channels 1 and 2 for the brightest antibodies
and the far red channels for lower expression staining.


Regards.


Vladislav Rozenkov, M.D., Ph.D


-----Original Message-----
From: Tracy Fischer-Smith <tlfsmith@temple.edu>
To: cyto-inbox
Sent: Thu, 22 Jun 2006 09:04:32 -0400
Subject: macrophage autofluorescence

Hello,


I have significant auto-fluorescence from cultured/infected/cytokine treated
monocytes/macrophages that inhibits my ability to see anything by flow.  I
have seen some methods for quenching macrophage auto-fluorescence on the web
and was hoping that if any of you have had experience with any of these
methods you wouldn?t mind to send me a quick response regarding what
method(s) you find to be most effective.  Also, would the use of a quenching
dye, such as crystal violet or trypan blue affect subsequent intracellular
staining?


Thanks in advance!


Tracy Fischer-Smith, PhD

Temple University

Dept. of Neuroscience

Center for Neurovirology

Philadelphia, PA