GFP compensation

Nathan Regimbal Nathan.Regimbal at biogenidec.com
Mon Mar 8 15:11:51 EST 2004


Dear Rama,

GFP and FITC are a bit different, so you can't really compensate for GFP 
using FITC.  You state that you're looking for +/- regulation of surface 
markers in the GFP + cells.  If you've only got a 'few' GFP positive cells 
(not enough to set compensation), then it doesn't matter if compensation 
is set right or not because the data's not there.  You'll need thousands 
of events for MFI stats for upregulation analyses.  For frequency 
(percentage positive) analysis you won't need as many cells but it is 
important that a) compensation is either set properly if you're using 
quadrants or b) you're crafty with regions and uncompensated data. 
Whatever the case, DON'T OVERCOMPENSATE the GFP. 

To eliminate the need to compensate GFP out of FL2, see if you can use a 
stain like PerCP or APC for detection of the surface marker on a 'Calibur. 
 GFP overlap in those channels is much less than in FL2, so compensation 
won't need to be needed too much to see a shift or count positive cells. 
Frankly, I'm not really stoked about analyzing the PE expression of 
GFP-positive cells.  GFP just tails up into FL2 too much.  Any upwards 
shift in GFP forces you to question if any upwards shift in FL2-ness was 
caused by increasing FL2 signal or just the concomitant increase in FL2 
'overlap'.

Don't think Ninny...think FlowJock.

Nate





"rama akondy" <rama_akondy at yahoo.com> 
03/08/2004 04:53 AM
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Subject
GFP compensation






dear Flow gurus,
 Being a bit of a ninny w.r.t flowcytometry ,need some
help. I am trying to look at up/down regulation of
certain surface markers in transiently trasfected
eGFP+ cells (FACScalibur).I use class-I staining (on
FITC on the same cells) for setting FITC/PE
compensations , the problem is that the eGFP+ cells
(but not the FITC+ controls) look like theyve been
over compensated, everything goes and sticks to the
axis. the proportion of GFP+s cells is so low that i
am not sure i shd rely on them to actually set
compensations..one would expect FITC and GFP to behave
similarly wudnt one?

Please enlighten
rama

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