GFP-PI

david.mcfarland@mcmail.vanderbilt.edu david.mcfarland at mcmail.vanderbilt.edu
Wed Feb 11 10:57:08 EST 1998


I have a client who is transfecting with GFP and then staining with PI and using
the subdiploid peak method to study apoptosis.  I'm operating a FACSCalibur and
have a few questions/difficulties.  Any suggestions would be appreciated.  He is
fixing the cells in 5%(?)paraformaldehyde and then storing overnight in 70%
ethanol.

1)  Can I do compensation between FL1(log) and FL2-Area(lin)?  The FL2
fluorescence contribution from the GFP doesn't seem to be significant, but... 

2)  The peaks are very broad.  What can he do to decrease cvs?

3)  We're seeing a peak off scale at the low end that would normally consist of
debris.  Gating first on forward scatter and drawing a gate around the
population of intact cells usually gets rid of most of this.  However, in this
instance it did not.  In addition, back gating on this "peak" shows that
everything in this region is GFP+ and they also come up on the forward vs side
scatter plot in the area where intact cells should be.  I should also mention
that we don't really see a subdiploid apoptotic peak where expected.  I came up
with a few scenarios to explain this.  Do any of these sound likely?  Theory1: 
His time point is too late and the "peak" is very late apoptosis.  Theory 2: 
Apoptotic bodies?  Could they be big enough to be included with intact cells
based on light scatter?  Why would they ALL be GFP+?  Theory 3:  The PI just
isn't getting in.  Poor permeabilization could also explain the high cvs, no?

Recent postings have touched on some of these areas, but I could use specific
answers to specific questions.  If you have an answer, I'd like to hear it. 
Thank you much.

David McFarland
Howard Hughes Medical Institute
Flow Cytometry Facility
Vanderbilt University Medical Center     




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