From: Ann Atzberger (Ann.Atzberger@EMBL-Heidelberg.de)
Date: Thu Dec 09 1999 - 08:31:37 EST
Hello everybody, at present I'm having a problem with CFP (Golgi fusion) expressing HeLa cells. In the initial round of sorting I can detect the cells above background without a problem, excitation is at 457nm em515/10 (I know I need a better em filter here; working on it). I can purify them to approx 60-80% (depending on how stable they are) in the first round of sorting; however in the 2nd round the CFP signal appears to have diminished - i.e few cells above background- (maybe in the transient phase the cells are brighter). Under the microscope one sees high (blueish) autofluorescence but also the CFP signal i.e approx 60%pos. - not very bright but there. So what I was thinking of doing is: staining the cells with CFDA, to interfere with the background fluorescence, which isn't excited at 457nm. Right? Will it work?? Why not? Regards Ann PS: We can't afford a 433nm laser (which would be the ideal excitation) because the users have just bought me a MoFlo, and anyway I would have to give priority to a new UV laser, the one I have being 12 years old and decrepit, and on its last legs. I've the wake already planned.
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