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 conjunction with various combinations of three Aequorea victoria green fluorescent protein (GFP) variants--EYFP, EGFP, and ECFP. In spite of overlapping emission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generated fluorescence signals that could be electronically compensated in real time using dual-laser excitation at 458 and 568 nm. Resolution of fluorescence signals from DsRed, EYFP, and EGFP was also readily achieved by single-laser excitation at 488 nm. Since many flow cytometers are equipped with an argon-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 and expression in mammalian cells. The dual-laser technique is applicable for use on flow cytometers equipped with tunable multiline argon-ion and krypton-ion lasers, providing the framework for studies requiring simultaneous analysis of four fluorescent gene products within living cells.
Hawley
T S
TS
Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, MD, USA.
Telford
W G
WG
Ramezani
A
A
Hawley
R G
RG
D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D014644:Genetic Variation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007834:Lasers; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D012190:Retroviridae / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D014162:Transfection; D000097573:Red Fluorescent Protein
D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C054562:yellow fluorescent protein, Bacteria