Simultaneous measurement of two cellular antigens and DNA using fluorescein‐isothiocyanate, R‐phycoerythrin, and propidium iodide on a standard FACScan

Abstract

Multiparameter flow cytometry is a powerful tool for analyzing the phenotypic, cell kinetic, and ploidy heterogeneity of tumor cell populations. 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 standard flow cytometer for these kind of studies. Instead of PI, 7-amino-actinomycin D (7-AAD) is used as DNA stain. In this paper however, we show 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 lysolecithin for the simultaneous detection of surface antigens by monoclonal antibodies MOv18, BMA180 or OV632, intermediate filament antigens (keratin 18 or vimentin), and DNA. A final concentration of 80 μg/ml lysolecithin was found to give optimal results. The emission spectrum overlap from PI into the orange fluorescence channel (FL2) used for 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, 5.10×10⁴ PE equivalents were detectable. Under these conditions, CVs obtained for the DNA histograms ranged from 3.0–4.1. Application of the method on a mixture 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.