DUAL-COLOR FLOW CYTOMETRIC DETECTION OF FLUORESCENT PROTEINS USING SINGLE-LASER (488-NM) EXCITATION

The ability to analyze independently the expression of multiple report er gene constructs within single cells is a potentially powerful appli cation of flow cytometry, In this paper, we explore the simultaneous d etection of two variants of the reporter molecule, green fluorescent p rotein (GFP) that both fluoresce when excited with 488-nm light, One o f these, enhanced GFP (EGFP) (excitation max, 490 nm; >90% efficiency at 488 nm), has been widely used for studies that involve how cytometr ic detection of reporter gene expression, As a partner for EGFP, we em ployed a recently described variant termed enhanced yellow fluorescent protein (EYFP) (excitation max. 513 nm; approximate to 35% efficiency at 488 nm), Using 488-nm excitation, EYFP fluorescence could be readi ly detected following expression of the gene in murine fibroblasts and this signal was comparable in intensity to that obtained from EGFP, I mportantly, we describe an optical filter configuration that permits t he fluorescence signals from both proteins to be distinguished by flow cytometry, despite their similar emission maxima. This filter configu ration employed a 510/20-nm bandpass filter for EGFP detection, a 550/ 30-nm bandpass filter for EYFP detection, and a 525-nm short-pass dich roic mirror to separate the two signals, With these filters, expressio n of either reporter protein could be detected, alone or in combinatio n, within a mixed population of cells over a broad range of signal int ensities. (C) 1998 Wiley-Liss, Inc.