Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins

Flow cytometric procedures are described to detect a "humanized" version of a new red fluorescent protein (DsRed) from the coral Discosoma sp. in conj unction with various combinations of three Aequorea victoria green fluoresc ent protein (GFP) variants-EYFP, EGFP, and ECFP. In spite of overlapping em ission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generate d fluorescence signals that could be electronically compensated in real tim e using dual-laser excitation at 458 and 568 nm. Resolution of fluorescence signals from DsRed, EYFP, and EGFP was also readily achieved by single-las er excitation at 488 nm. Since many flow cytometers are equipped with an ar gon-ion laser that can be tuned to 488 nm, the DsRed/EYFP/EGFP combination is expected to have broad utility for facile monitoring of gene transfer an d expression in mammalian cells. The dual-laser technique is applicable for use on flow cytometers equipped with tunable multiline argon-ion and krypt on-ion lasers, providing the framework for studies requiring simultaneous a nalysis of four fluorescent gene products within living cells.