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

Evaluation of enhanced green fluorescent protein (EGFP) for use in gene exp ression studies was done by transfection of several EGFP variants into mamm alian cell lines followed by measurement of fluorescence intensities using a microplate reader. Fluorescent excitation and emission spectra of EGFP ex pressing living cells were arithmetically folded with excitation and emissi on filter transmission data allowing the calculation of fluorescence yields for different filter combinations. FITC (fluorescein-5-isothiocyanate) fil ter 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 nea rly linear dependence of fluorescence on cell numbers was observed, the low er limit of detection being 11,400 adherent cells per well (96-well plate). For strong EGFP expression of transiently transfected cells >7% positive c ells in a confluent cell lawn can be distinguished from nonfluorescent cont rols (24-well plate). For weak EGFP expression from inducible promoters it is of great importance to consider the background from cells and media comp onents. The use of balanced salt solution instead of media and special micr oplates designed for fluorescence readings of mammalian cells can reduce th e contribution of background fluorescence. For kinetic measurements which c onsider the modulation of gene expression (up/down regulation) and which re sult in less fluorescence recorded, especially when d2EGFP is used, the abo ve mentioned constraints have to be more strictly inspected. (C) 2001 Elsev ier Science B.V. All rights reserved.