Simultaneous analysis of the cyan, yellow and green fluorescent proteins by flow cytometry using single-laser excitation at 458 nm

Background: Development of spectrally distinct green fluorescent protein (G FP) variants has allowed for simultaneous now cytometric detection of two d ifferent colored mutants expressed in a single cell. However, the dual-lase r methods employed in such experiments are not widely applicable since they require a specific, expensive laser, and single-laser analysis at 488 nm e xhibits considerable spectral overlap. The purpose of this work was to eval uate detection of enhanced cyan fluorescent protein (ECFP) in combination w ith the enhanced green (EGFP) and enhanced yellow (EYFP) fluorescent protei ns by now cytometry. Methods: Cells transfected with expression constructs for EGFP, EYFP, or EC FP were analyzed by now cytometry using excitation wavelengths at 458, 488, or 514 nm. Fluorescence signals were separated with a custom optical filte r configuration: 525 nm shortpass and 500 nm longpass dichroics; 480/30 (EC FP), 510/20 (EGFP) and 550/30 (EYFP) bandpasses; 458 nm laser blocking filt ers. Results: All three fluorescent proteins when expressed individually or in c ombination in living cells were excited by the 458 nm laser line and their corresponding signals could be electronically compensated in real time. Conclusions: This method demonstrates the detection of three fluorescent pr oteins expressed simultaneously in living cells using single laser excitati on and is applicable for use on flow cytometers equipped with a tunable arg on ion laser. (C) 1999 Wiley-Liss,Inc.