RE: autofluorescence and compensation

the cell type is irrelevant for compensation.	If you get better staining with fewer dead cells and (most important) the SAME brightness of fluorescence, go ahead and use PBL or mouse spleen. Or invest in Ab capture beads.

To amend your current approach, you should use PI (or another viability stain) to gate out the dead cells. Then set your compensation.	 If you are doing analysis, not sorting, you can save yourself a great deal of time by collecting your comp samples and experimental samples with all data uncomp, then using FlowJo to perform softward compensation

please see www.drmr.com/compensation/ for Mario's excellent tutorial thru the principles and practices of compensation

=======================================================
Rachel M. Gerstein, Ph.D.
Associate 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) 



-----Original Message-----
From: Eric Shaw [mailto:eseric47@googlemail.com]
Sent: Wed 6/11/2008 12:20 PM
To: cyto-inbox
Subject: autofluorescence and compensation
 
Hi Flowers,
I have a question.
I have a tissue dissagregation which will have lots of dead cells and
various cell types which most likely have different amounts of
autofluorescence. I know I need to use single stained controls (unstained,
7AAD, FITC,PE,APC) but wont my compensation be inaccurate for FITC/PE/APC
since I am compensating for the whole population- live and dead? And what
about the different cell types (epi's, tissue macrophages etc) that can give
differing amounts of autoflorescence? I compensate with my R1 gate  off
FSC/SSC and I am using a FACS Calibur. All replies will be appreciated.

Thanks,

Eric Shaw