Simultaneous Analysis of Three Fluorescent Proteins (Cyan, Green and Yellow) Using Single-Laser Flow Cytometry
    The expression of the green fluorescent protein in mammalian systems is a useful tool for the study of a variety of cellular functions and processes. The recent introduction of the spectral varants of the EGFP (GREEN) such as ECFP (CYAN) and  EYFP (YELLOW) now provide for the simultaneous analysis of three fluorescent proteins. This three-colour analysis can be performed using a single excitation wavelength at 458 nm from an argon-ion laser on a standard flow cytometer (Beavis AJ and Kalejta RF, Cytometry 37:68-73, 1999) and is therefore advantageous compared to the dual-colour, dual-laser methods previously described.
    As shown below in the two-colour dot-plots, the simultaneous expression of EGFP, EYFP and ECFP can be quantitated and the 8 possible subpopulations defined using multicolour gating:


 

Primary human cells were transfected with expression plasmids for EGFP, EYFP and ECFP, cultured and analysed for fluorescence. Data are displayed as three dual-parameter plots EGFP/EYFP (A), EGFP/ECFP (B) and EYFP/ECFP (C). Fluorescence emissions were separated using a custom optical configuration. 
Boolean gate logic and multicolour gating can be used to identify the 8 possible subpopulations:		 BLACK  = triple negatives 
GREEN  = GFP+ single positives 
CYAN   = CFP+ single positives 
YELLOW  = YFP+ single positives 
ORANGE  = GFP+/YFP+ double positives 
MAGENTA  = GFP+/CFP+ double positives 
DARK BLUE = CFP+/YFP+ double positives 
RED   =  GFP+/YFP+/CFP+ triple positives
 
Full details can be found in:
Beavis AJ  and Kalejta RF. Simultaneous analysis of the cyan, yellow and green fluorescent proteins by flow cytometry using single-laser excitation at 458 nm. Cytometry 37(1), pp 68-73. 1999.
Andrew Beavis,
Manager, Flow Cytometry Core Facility,
Dept. Molecular Biology,
Princeton University, Princeton NJ 08544
Tel:(609) 258 1695
email:Abeavis@molbio.princeon.edu
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