Fluoro-gold: An alternative viability stain for multicolor plow cytometricanalysis

Background: The viability stains propidium iodide (PT) and 7-amino-actinomy cin D (7-AAD) are excited at 488 nm, as are the commonly used antibody conj ugates fluorescein isothiocyanate (FITC), phycoerythrin (PE), and cyanine 5 dye covalently coupled to R-phycoerythrin (RPE-Cy5). When excited by a sin gle laser, spectral overlap in the emission of PI and 7-AAD with RPE-Cy5 pr ecludes the use of these viability stains for three-color immunophenotyping , particularly when evaluating low levels of marker expression in viable ta rget cells. The ultraviolet excitable dye hydroxystilbamidine methanesulfon ate (Fluoro-Gold, or FG) binds to DNA at the A-T-rich regions of the minor groove in permeabilized or dead cells. We assessed the suitability of this dye as a viability stain. Methods: The ability of FG to detect nonviable cells in fresh and cryoprese rved human apheresed peripheral blood cells was compared with that of PI an d 7-AAD. The stability of FG staining and the effects of dye and cell conce ntration on the discrimination of nonviable cells was determined by measuri ng changes in the median fluorescence of viable and nonviable cells. Results: FG labeling at dye concentrations of 2-8 mu M is stable for at lea st 3 h over a wide range of cell concentrations (4 x 10(5) to 4 x 10(7) cel ls/ml). Costaining studies and linear regression analysis show that cell vi ability as determined by FG is strongly correlated with estimates using PI (r = 0.9636) and 7-AAD (r = 0.9879). Conclusions: FG is a reliable, alternative viability stain that can be used in conjunction with fluorochromes including FITC, PE, and RPE-Cy5 for mult icolor analysis using dual-laser instruments. (C) 1999 Wiley-Liss, Inc.