Three-color flow cytometry analysis of tricistronic expression of eBFP, eGFP, and eYFP using EMCV-IRES linkages

Background: The ability to quickly analyze and sort double or triple fluore scent reporter constructs using simultaneous analysis provides significant flexibility in the solution of analytical and process-related questions in biotechnology. Methods: Bicistronic eBFP/eGFP and eBFP/eYFP constructs were made on two ma mmalian episomal plasmids using an internal ribosomal entry sequence from e ncephalomyocarditis virus (EMCV-IRES) to link two GFP expressions. Simultan eous two-color flow cytometry (FCM) analysis was accomplished using a dual Argon-laser multi-line configuration set at excitation wavelengths of 360 a nd 488 nm. Blue fluorescence emission (440 nm) and green fluorescence emiss ion (507 nm) were detected using 405/20 (FL4) and 510/20 (FL1) bandpass fil ters. Dual eBFP/eYFP and three-color simultaneous analysis of eBFP/eGFP/eYF P was accomplished using the dual-laser configuration brit also using a sho rt-pass (525-nm) dichroic mirror and 550/30 bandpass filter configuration t o detect yellow fluorescence emission (527 nm) in a third channel (FL2). Results:: Human 293 cells transfected with the bicistronic construct of eBF P-IRES-eGFP were easily detected using simultaneous analysis, and the signa ls were well separated with a mean blue fluorescent intensity (MFI) in the 2nd-log decade (FL4) and green MFI in the 4th-log decade (FL1). Likewise, e BFP-IRES-eYFP transfected cells were as easily detected and also demonstrat ed very good signal separation. A tricistronic construct of eBFP-IRES-eGFP- IRES-eYFP was also made and transfected into 293 cells. Triple-color fluore scent cells mere easily detected using the cytometer configuration for simu ltaneous analysis. All three signals separated with only moderate compensat ion required for green and yellow emission spectra. The respective MFI for each of the fluorescent proteins was correlative to what had been observed with the separate bicistronic constructs. Conclusions: Our results demonstrate that we have developed a novel fluores cent flow cytometry method that can be used as a powerful tool to different iate and analyze three colors simultaneously from either a dual or a triple cistronic construct which has been transfected into living cells. (C) 1999 Wiley-Liss, Inc.