The excitation wavelength of the original GFP from jellyfish (wtGFP) was not really suitable for 488 nm laser. Also, jellyfish codon usage was not efficiently translated by mammalian cells. I remember there was also some discussion on temperature sensitivity. But now there are a lot of commercially available GFP mutants which were actually selected for detection by flow cytometry. I have used pGreenLantern-1 from GIBCO BRL and pEGFP-1 from CLONTECH, and both worked very well in flow. I compared pGreenLantern-1 with wtGFP in the same cell line, and the difference in the mean fluorescence intensity was 2 to 3 log's. Mayumi Naramura, M.D. NIH/NIAID Lab of Immunology Twinbrook II, Rm. 125 12441 Parklawn Drive Rockville, MD 20852 Phone: 301-402-4595 FAX: 301-594-2522 e-mail:mnaramura@atlas.niaid.nih.gov ---------- From: David L. Haviland, Ph.D. Sent: Tuesday, February 25, 1997 11:29 To: cyto-inbox Subject: GFP Hi: I was curious if people had been successfully analysed GFP levels within transfected cells using flow? I once had an investigator try something like this (though I do *not* know if it was GFP, per se) and the experiment was a complete bust! There was something about the excitation/emmision that was not near instantaneous and there was enough delay that no fluoresence could be detected except when using a flourescent microscope. David
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