Transfection efficiency by GFP - (May/08/2006 )

Hi everybody!

I want to check the effect of my protein on cell survival. First I tried to make stable cells but this didn´t work twice (!!!)- the cells were expressing really low amount of my protein.

Now I decided to do this in transient transfection but I don´t know how to monitor the efficiency of transfection. I heard one can use GFP for that. Is it possible to do it without using FACS but simply measuring the intensity of GFP from the well (Im planning to do it in 96 well plates). What kind of spectrophotometer would I need for that?

Thanks,

Kamila

I think using the FACS or a fluoresence microscope (less sensitive than FACS) would be a good idea. You get a better idea of the % positive cells. Otherwise you measuring whether you have some transfected cells versus no transfected and you don't know if you have a few high expressing cells or a lot of low/medium expressing cells. The FACS should give you this quantitiation. If you looking at effects on surface markers or a protein you can measure by intracellular FACS you could do two colour FACS also.

Good luck,
Ceri

FACS might be a better way to quantify your transfections than spectrophotometer. Or just count the fluorescent cells in a 40x field after transfection, would give a rough estimate.

Another way to measure transfection efficiency is to use beta-gal. You can transfect in the plasmid and use a reagent that will induce fluorescence in transfected cells. this has been used with FACS and is the basic principle behind luciferase assays. You could also use a luciferace plasmid.

I think if you are going to measure efficiency with GFP, you need to use FACS or the fluorescent microscope. I think it would be possible to us the spectrophotometer, but I would be very critical of the outcome.

I found EGFP-luciferase fusion vector from clontech (Now BD Biosci) serve me well for % cell positive for GFP as well as absolute number of light units/well. Hope that help.