AEQUOREA GREEN FLUORESCENT PROTEIN-ANALYSIS BY FLOW-CYTOMETRY

The isolation and expression of the cDNA for the green fluorescent pro tein (GFP) from the bioluminescent jellyfish Aequorea victoria has hig hlighted its potential use as a marker for gene expression in a variet y of cell types (Chalfie et al.: Science 263: 802-805, 1994), The long er wavelength peak (470 nm) of GFP's bimodal absorption spectrum bette r matches standard fluorescein filter sets; however, it has a consider ably lower amplitude than the major absorption peak at 395, In an effo rt to increase the sensitivity of GFP with routinely available instrum entation, Heim et al, (Nature 373:663-664, 1995) have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single abs orption peak centered at 490 nm. We have constructed this mutant in or der to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima, Usi ng the conventionally available 488 nm and ultraviolet (UV) laser Line s from the argon ion laser as well as the 407 nm line from a krypton i on laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry, The highest fluorescence si gnal was observed with 488 nm excitation of S65T-GFP relative to all o ther laser line/GFP pairs, The wt-GFP fluorescence intensity, in contr ast, was significantly higher at 407 nm relative to either 488 nm or U V, These results were consistent with parallel spectrofluorometric ana lysis of the emission spectrum for wt-GFP and S65T-GFP. The relative c ontribution of cellular autofluorescence at each wavelength was also i nvestigated and shown to be significantly reduced at 407 nm relative t o either UV or 488 nm. (C) 1995 Wiley-Liss, Inc.