FGP AND CELL CYCLE???

Derek Davies daviesd2 at icrf.icnet.uk
Mon Jul 9 01:27:44 EST 2001


Hi Tim,

This is a question that often arises when workers are using FGP as a
marker of transfection. The commonest FP is GFP but a slight problem
with this is that the fluorescence is usually lost after ethanol
fixation which is optimal if you are also using PI to get cell cycle
profiles. There are a number of ways round this:

1. Use E-GFP-F (from Clontech) which has a farnesylated protein that
localises in the plasma membrane - fluorescence is then preserved after
ethanol fixation. This is the best approach for new projects and for
being able to do on a standard bench-top flow cytometer but this may
not be possible if you already have a fusion protein system working.

2. Try a sequential fixation of 1% paraformaldehyde then alcohol
(optimal times and concentrations will vary and should be determined).
The cross-linking aldehyde fixation preserves the GFP fluorescence and
the alcohol allows a more precise DNA histogram.

3. Use Hoechst 33342 as a vital DNA dye which gets away from the
fixation problems but requires a UV laser (and would be difficult if you
are using BFP). This is the approach we have taken, initially on a cell
sorter but recently on the BD LSR which has a HeCd UV source. We get
good DNA CVs and as the cells are live we still see strong GFP
expression - you can then gate on the positive GFP cells and get a DNA
profile. As the cells are live you can also use a dead cell
discriminatory dye (PI, 7AAD, TP3) to exclude dead cells from the
analysis.

4. Sort the FP positive cells, ethanol fix and run a standard DNA
analysis. A bit long-winded if you have a lot of samples though.

5. Not a flow method, but you could also use a Laser Scanning Cytometer.
This would allow sequential analysis of cells so you could measure GFP
fluorescence, fix the cells in situ, do PI analysis for cell cycle and
then, as positional information is recorded, merge the files.

Hope that is of some help!

Derek


On Thu, 5 Jul 2001, Tim Kute wrote:
>         A fellow investigator has asked if the expression of FGP
> (fluorescent green protein) is cell cycle related?  Are there any flow
> people that have tried to answer this question.  If so, what was the
> results and how did you do it by flow?

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Derek Davies                       Voice: (44) 020 7269 3394
FACS Laboratory,                   FAX: (44) 020 7269 3100
Imperial Cancer Research Fund,     e_mail: derek.davies at icrf.icnet.uk
London, UK			   mobile: 07790 604112

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