Compensating cultured cells Dennis_Young at
Fri Mar 15 19:46:00 EST 1996

Changing the type of media will change autofluorecence dramatically (I think 
DMEM gave the most AF!). Soaking in plain PBS will help leach out the vitamins 
that are the main culprates. I didn't get any help on how to use borohydride 
from this BB, but there was a paper (Beisker,W, Dolbeare,F, Gray, JW, 

* Dennis J. Young                            Voice : (619) 822-0407         *
* Flow Cytometry Core Facility               FAX   : (619) 822-0412         *
* University of California, San Diego  USA   e-mail: djyoung at       *
______________________________ Reply Separator _________________________________
Subject: Re: Compensating cultured cells
Author:  Roederer at Beadle.Stanford.EDU at @UCSD
Date:    3/15/96 12:06 PM

>   Occasionally, I need to run FACS on cultured cells (bone
>   marrow, specifically). I am challenged ad nauseum, at
>   achieving a readable analysis as I cannot seem to
>   compensate-out the slant I seem to always get. I would
>   like to hear of any hints anyone might have in dealing
>   with cells that present in this manner. I use a B-D
>   Facscan.

The problem you are seeing is probably due to autofluorescence.  Cultured cells
tend to have significantly increased autofluorescence.  The fluorescence
spectrum of AF is such that the signal is highly correlated in (essentially) all
channels.  Since the spectrum is distinct from fluorescein, PE, etc., proper
compensation of these fluors will leave autofluorescent cells as having a
diagonal characteristic.

There is little to be done about this; if you have a channel to spare, you can
do autofluorescence compensation.  I.e., if you are measuring only FITC, then
use the PE to compensate the autofluorescence in the FITC channel (as well as
the Cy5PE channel; then you can do 2-color immunofluorescence with AF
compensation).  However, if you don't have a spare channel, or if you need to do
PE staining, then you won't get rid of the "diagonal".


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