cell cycle analysis of transiently transfected cells - (Jun/23/2005 )

Hi all. another cell cycle analysis question but this involves using a transfected cell line.

I am wishing to do CCA on transfected cells expressing a EGFP reporter and my gene of interest, which i believe inhibits p53 leading to an increase in the number of cells in S-phase

I am using a liver stem cell derived line that only supports between 30-45% transfection, as confirmed by UV microscopy. My problem is that when I run my cells through FACS, there is virtually no EGFP cells present, and this is a problem as I wish to a DNA content analysis (by PI staining) of ONLY those cells expressing EGFP ie: containing my gene of interest.

fixing with 70% ethanol appears to not help the problem
also fixing in 2% PFA prior to using 70% ethanol does not help either. My problem is I think that the EGFP expressing cells are weakened and are lysed by the ethanol.
To my knowledge, in comparison to UV microscopy, FACS is a much more sensitive method thay should pick up more EGFP + cells than I can actually count underneath the mircoscope. However I have not seen this yet.

in addition, I think that the actual trypsinisation process when i harvest the cells may be comprimising the membrane leading to the leaking out of the EGFP contributing to the low number of EGFP + cells that I am seeing during.

Should i try a just EDTA method method to get cell detachment, with some mechanical manipulation to get the cells to detach, which will hopefully prevent membrane being comprimised?

and while I'm on the topic. WHat software is best to use? I am currently using FLowJo 6.2 cell cycle parameter but I am finding it difficult to fit algorithms to my data. is there any good freeware/shareware available for windows XP?

any help would be greatly appreciated as this is is crucial to my honours project

ps: apologies for the lengthy post

Hi.

70% ethanol will cause the EGFP to be lost because it permeablizes the cell membrane. One thing you might try is "live" cell cycle analysis using Hoechst 33258 dye. You won't need to fix your cells in order to to measure DNA content.

The only thing is that you will have to do some compensation when setting up your flow cytometer settings because Hoechst 33258 does bleed over into the EGFP channel. Plus, I think it needs to be done on a dual-laser machine, b/c Hoechst 33258 abosrbs in the UV range. I hope this helps! Good luck!!!