SIMULTANEOUS MEASUREMENT OF 2 CELLULAR ANTIGENS AND DNA USING FLUORESCEIN-ISOTHIOCYANATE, R-PHYCOERYTHRIN, AND PROPIDIUM IODINE ON A STANDARD FACSCAN

Multiparameter flow cytometry is a powerful tool for analyzing the phe notypic, cell kinetic, and ploidy heterogeneity of tumor cell populati ons. Because of the substantial spectral overlap of propidium iodide ( PI) and R-phycoerythrin (PE) fluorescence emission, this combined use of these fluorochromes has been thought not to be feasible on a standa rd flow cytometer for these kind of studies. Instead of PI, 7-amino-ac tinomycin D (7-AAD) is used as DNA stain. In this paper however, we sh ow that PI can be used as a DNA stain in combination with fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE) on a standard FACScan. Three established ovarian cancer cell lines (IGROV1, NIH: OVCAR-3, and COV362.c14) were used for these experiments. Cells were fixed with 1. 0% paraformaldehyde and permeabilized with various concentrations of l ysolecithin for the simultaneous detection of surface antigens by mono clonal antibodies MOv18, BMA180 or OV632, intermediate filament antige ns (keratin 18 or vimentin), and DNA. A final concentration of 80 mu g /ml lysolecithin was found to give optimal results. The emission spect rum overlap from PI into the orange fluorescence channel (FL2) used fo r PE fluorescence detection could be sufficiently compensated up to a photomultiplier tube potential of about 440 Volts (V) required at the FL2 channel. Using the same instrument settings, 5.10 x 10(4) PE equiv alents were detectable. Under these conditions, CVs obtained for the D NA histograms ranged from 3.0-4.1. Application of the method on a mixt ure of activated peripheral blood lymphocytes and ovarian tumor cells resulted in a clear separation of the two populations both by surface and cytoplasmic antigen expression and DNA content. (C) 1994 Wiley-Lis s, Inc.