From: Maggie Kasten (kasten1@jeflin.tju.edu)
Date: Tue Sep 15 1998 - 08:34:58 EST
Dear Flow-ers, We've started working with a variant of EGFP, called EGFP-F, that is targeted to the cell membrane when transfected into mammalian cells. It was originally created to eliminate the problem of losing GFP signal when fixing cells with ethanol (W. Jiang and T. Hunter, March 1998 Biotechniques 24:348-354). Since we want to stain our cells with propidium iodide at the same time, ethanol fixation seemed to be a good choice. However we've run into a different problem with this construct. When we fix trypsinized EGFP-F transfected cells with 70% ethanol and examine them using a fluorescence microscope we find that _all_ the cells show some fluorescence. There are a smaller number of cells that fluoresce very brightly, which are presumably the true original transfected cells, but the background is quite high. The weaker fluorescing cells seem to have small patches of brightness associated with them as opposed to having signal throughout the cell membrane. Unfixed EGFP-F transfected cells have a bright fluorescent population and the other cells remain completely dark/blank. Therefore, this background seems to be a problem associated with our fixation procedure. The background does not appear in the untransfected cell line or in cells transfected with the more traditional cytosolic EGFP construct. As you would expect from these results, when the fixed EGFP-F transfected cells are examined by flow cytometry, the entire "background" peak of fluorescence shifts from a very low fluorescent signal indicating a higher fluorescent background. The shift is so extreme that it makes it impractical to use this method to identify the transfected population by flow cytometry. We are going to try other fixation protocols to try to resolve this problem, but I wanted to know if there are other researchers (besides the original authors) who have used this construct. Have you encountered this phenomenon? If so, have you determined a fixation protocol that eliminates this problem? We have observed this phenomenon in one adherent cell line and in one suspension cell line. Thanks for any help. Maggie Kasten -- Maggie Kasten Voice: 215-503-4614 Pathology Department FAX: 215-923-9626 Thomas Jefferson University Philadelphia, PA kasten1@jeflin.tju.edu
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