Suitability of enhanced green fluorescent protein as a reporter component for bioassays

Abstract

Evaluation of enhanced green fluorescent protein (EGFP) for use in gene expression studies was done by transfection of several EGFP variants into mammalian cell lines followed by measurement of fluorescence intensities using a microplate reader. Fluorescent excitation and emission spectra of EGFP expressing living cells were arithmetically folded with excitation and emission filter transmission data allowing the calculation of fluorescence yields for different filter combinations. FITC (fluorescein-5-isothiocyanate) filter sets used for EGFP measurement do not meet the EGFP optimum. Microplate readers equipped with custom made filters may lead to higher fluorescence readings, but to exclude the contribution of excitation wavelengths in the emission wavelength range of EGFP excitation filters with a maximum at 460–470 nm instead of 480–490 nm should be used for red-shifted EGFP variants. For stably transfected cell lines where nearly all cells express EGFP a nearly linear dependence of fluorescence on cell numbers was observed, the lower limit of detection being 11,400 adherent cells per well (96-well plate). For strong EGFP expression of transiently transfected cells >7% positive cells in a confluent cell lawn can be distinguished from nonfluorescent controls (24-well plate). For weak EGFP expression from inducible promoters it is of great importance to consider the background from cells and media components. The use of balanced salt solution instead of media and special microplates designed for fluorescence readings of mammalian cells can reduce the contribution of background fluorescence. For kinetic measurements which consider the modulation of gene expression (up/down regulation) and which result in less fluorescence recorded, especially when d2EGFP is used, the above mentioned constraints have to be more strictly inspected.