transfection gfp visualization - (Jun/26/2006 )
i am going to visualize GFP constructs for the first time...my transfection is not working at all and so i thot of using this approach to get some idea.
can anybody tell me the protocol for visualizing the cells on coverslips or directly in TC plate?? what all instruments can we use ?
i am using lipo 2000 and transfecting HeLa cells. I usually remove the complex after 4-5 hrs and add DMEM +FCS (tried various conc i.e..1%, 2%, 5% and 10%) but get a very high background signal in untransfected cells too. and in my positive control (SEAP assay, ClonTech).
would appreciate any help/comments/advise.
Are you using a fluorecence microscope to view EGFP+ cells? If you see high background, then you did not transfect hela cells sucessfully, because otherwise you would see distict bright green cells.
I typically use plate method. I replace transfection mixture with 10% FBS-DMEM post transfection.
for the question of coverslip and dish... I was taught that coverslip is better for visualization because the plastic dishes give autofluorescence. If a confocal picture is needed, coverslip is much better. But for visualization of expression of GFP, both can be used.
I have noticed that the condensation on the cover of the tissue culture plates is related to the increased background if inverse microccope is used. Change the original warm medium to the one that is at room temperature, and minimizing observation time help to prevent such condensation.
There is some kind of autofluorescence-reduce medium, which I used for life-time imaging, gives less background than DMEM or other "colorful" medium. But it only can be used temperarily, cells can not grow well there...... Anyway, If any one wants, I can try to find it back.