> 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. Ann, I would not use CFDA to quench the auto fluorescence because the excitation spectra of the CFDA (475-492nm at Acid->Alkali pH) is very close to the emission spectra of your CFP. FRET may occur and you may lose your CFP signal altogether. Also the filter set that you are looking at the CFP fluorescence is perfect for the emission of CFDA( 517nm) and would compound the problem if you use a 488nm line as well. The 457nm line that you excite CFP with will excite CFDA minimally, but that excitation may be enough to saturate your CFP signal. Hope this helps. Andy. Andy Riddell PNAC Division MRC LMB Hills Road Cambridge UK tel: (0)1223 402218 fax: (0)1223 412178 email ar3@mrc-lmb.cam.ac.uk
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