PI and GFP

Wendy D. Schober (wschober@bcm.tmc.edu)
Mon, 24 Feb 1997 10:34:51 -0600 (CST)

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@bcm.tmc.edu