HEK cell, EGFP and PI staning - (Feb/29/2008 )

Hi!!!

Is somebody can help me?

I transfected HEK293T cells with EGFP plasmids (pEGFP-N1 or pEGFP-C3 from Clontech) and I analysed the cell cycle on flow cytometry.
I had problem to detect EGFP when I used pEGFP-N1 plasmid and I detect a little when I used pEGFP-C3 while I saw green cells on microscopy!!

I think the problem is due to fixation of cells before PI staining.
I use a standard protocol with EtOH 70%.

Maybe this protocol is not adapted to my cells?
Is somebody already have a similar problem? Could you recommand me an other protocol?

Thanks for all!!!!!

Hi!!!

Is somebody can help me?

I transfected HEK293T cells with EGFP plasmids (pEGFP-N1 or pEGFP-C3 from Clontech) and I analysed the cell cycle on flow cytometry.
I had problem to detect EGFP when I used pEGFP-N1 plasmid and I detect a little when I used pEGFP-C3 while I saw green cells on microscopy!!

I think the problem is due to fixation of cells before PI staining.
I use a standard protocol with EtOH 70%.

Maybe this protocol is not adapted to my cells?
Is somebody already have a similar problem? Could you recommand me an other protocol?

Thanks for all!!!!!

QUOTE (Auror37 @ Feb 29 2008, 09:23 AM)

Hi!!!

Is somebody can help me?

I transfected HEK293T cells with EGFP plasmids (pEGFP-N1 or pEGFP-C3 from Clontech) and I analysed the cell cycle on flow cytometry.
I had problem to detect EGFP when I used pEGFP-N1 plasmid and I detect a little when I used pEGFP-C3 while I saw green cells on microscopy!!

I think the problem is due to fixation of cells before PI staining.
I use a standard protocol with EtOH 70%.

Maybe this protocol is not adapted to my cells?
Is somebody already have a similar problem? Could you recommand me an other protocol?

Thanks for all!!!!!

Why are you fixing before PI staining?

[/quote]

I fixe the cells because I don't make the flow cytometry analysis the same day than the cell proliferation arrest.
[/quote]

Ah ok. Well you can't use PI on fixed cells - they will all be positive.

I never used EtOH fixation, but doesn't that make your membranes leaky for the EGFP? (not sure, I always use formaldehyde in PBS)
Why are you staining with PI after fixation? Indeed all of them will be positive. If you do it before fixation only apoptotic and other cells with ruined membranes will be PI-positive (so you can put a negative gate on them). Just stain with PI, wash it away (maybe 2-3 times) and then fix your cells.

pEGFP-N1 vector is for construction of fusion proteins where the EGFP is tagged the C terminus of your gene of interest. So if you transfect empty pEGFP-N1 vector into your cells, it is normal that your cells do not give green florescent.