PI and GFP

Wendy D. Schober wschober at bcm.tmc.edu
Mon Feb 24 11:34:51 EST 1997

We are attempting to optimize the procedure to label transfection along 
with cell cycle in HeLa cells.  So far we have co-transfected with CD20 
and then labeled the surface with CD20-FITC.  We found 5-13% CD20 
positive with good PI patterns after the usual 70% ethanol fixation.  
We used the same cells and fixation procedure with GFP as the reporter and 
hoped to find similar results.  Unfortunately, there were few cells GFP 
positive and what may be there are of very low intensity, barely over 
background.  We are looking for suggestions for improving our GFP and keeping 
good PI patterns.  The GFP is from Invitrogen and they have been somewhat 
helpful, but have not done this with PI.  
Is fixation causing a problem??  If so, is there a better procedure which 
will permeabilize and fix to get both GFP and PI to work?? Ultimately, we 
want this to work in some fibroblast lines, but HeLa is our control.  

Thanks in advance for any hints.
Wendy Schober
wschober at bcm.tmc.edu

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