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FACS GFP leakage - (May/07/2007 )

Hi,

I want to check if a GFP-fused protein of mine is affecting COS cell cycle. What I want to do is transfect, and then stain with some DNA dye (e.g. PI). I then use FACS to analyse cell cycle stage of GFP positive cells. The problem is, or so I have been told, that when the membrane is permeabilized for PI staining the GFP leaks out. I know one option is first to sort cells, and then stain the two groups seperatley, but our FACS doesn't sort. Does fixing cells with paraformaldehyde before/instead of ethanol help? I know dyes like DRAQ5 can work, but they're a bit above the budget (at least those I've come across).

thanks alot

-beenyg-

QUOTE (beenyg @ May 7 2007, 07:38 PM)
Hi,

I want to check if a GFP-fused protein of mine is affecting COS cell cycle. What I want to do is transfect, and then stain with some DNA dye (e.g. PI). I then use FACS to analyse cell cycle stage of GFP positive cells. The problem is, or so I have been told, that when the membrane is permeabilized for PI staining the GFP leaks out. I know one option is first to sort cells, and then stain the two groups seperatley, but our FACS doesn't sort. Does fixing cells with paraformaldehyde before/instead of ethanol help? I know dyes like DRAQ5 can work, but they're a bit above the budget (at least those I've come across).

thanks alot


have you ever tried if really all GFP-fusion protein is washed out? It depends on total expression of GFP-fusion protein as well as where it is subcellular localized; I would give a trial...

-The Bearer-

I think fixing cell with paraformaldehyde before membrane permeabilization might help to fix intracellular proteins in their particular compartment. Not sure about ethanol though...might not be strong enough.
Hope it worked out for you.

-Lynnpanda-

Fix with paraformaldehyde first, wash it out well then fix with ethanol as normal. In my hands, PI quenches GFP, FITC etc. so use the minimum amount of PI you possibly can.

Or if you have a fancy machine with a laser producing the appropriate wavelength, use DAPI instead. Its exitation and emmission is far from GFP so it is much easier to interpret the result.

-microphobe-

Did you check that your cells cen be detected by cytometry without fixing? the level of GFP would be low in your cells.

-NTH-