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High GFP background on FACS - (Oct/12/2008 )

Hi

I am trying to do FACS using a GFP fusion protein and analyze its affects of cell cycle. I see very strong endogenous auto-fluorescence of my negative cells which makes it very difficult to verify the GFP signal. I was wondering if someone knows how to eliminate this background? Thank you.

Haki

-Haki-

QUOTE (Haki @ Oct 13 2008, 11:43 AM)
Hi

I am trying to do FACS using a GFP fusion protein and analyze its affects of cell cycle. I see very strong endogenous auto-fluorescence of my negative cells which makes it very difficult to verify the GFP signal. I was wondering if someone knows how to eliminate this background? Thank you.

Haki


hello which microscope are you using now? Some microscope could eliminate the backgroud.

I am studying plant biology. So I do a lot of things about plant cells.

Stone

-stone757-

Hi Stone

Thank you for the response. Actually I am doing FACS analysis using FACScan. In the FL microscope I don't have too much background but in the FACS I have too much of auto-fluorescence in my negative control.

Thanks
Haki

QUOTE (stone757 @ Oct 13 2008, 01:41 AM)
QUOTE (Haki @ Oct 13 2008, 11:43 AM)
Hi

I am trying to do FACS using a GFP fusion protein and analyze its affects of cell cycle. I see very strong endogenous auto-fluorescence of my negative cells which makes it very difficult to verify the GFP signal. I was wondering if someone knows how to eliminate this background? Thank you.

Haki


hello which microscope are you using now? Some microscope could eliminate the backgroud.

I am studying plant biology. So I do a lot of things about plant cells.

Stone

-Haki-

Is it possible to upload some of your plots?
If you see high background on your Facs but not fluorescence microscope, maybe it's your voltage that's been set wrong (i.e. too high)?

QUOTE (Haki @ Oct 13 2008, 01:17 PM)
Hi Stone

Thank you for the response. Actually I am doing FACS analysis using FACScan. In the FL microscope I don't have too much background but in the FACS I have too much of auto-fluorescence in my negative control.

Thanks
Haki

QUOTE (stone757 @ Oct 13 2008, 01:41 AM)
QUOTE (Haki @ Oct 13 2008, 11:43 AM)
Hi

I am trying to do FACS using a GFP fusion protein and analyze its affects of cell cycle. I see very strong endogenous auto-fluorescence of my negative cells which makes it very difficult to verify the GFP signal. I was wondering if someone knows how to eliminate this background? Thank you.

Haki


hello which microscope are you using now? Some microscope could eliminate the backgroud.

I am studying plant biology. So I do a lot of things about plant cells.

Stone


-vairus-