TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry

A new red emitting fluorophore, TO-PRO-3 iodide (TP3), which is best excited by an HeNe laser (633 nm), has been compared with propidium iodide (PI) for measuring relative DNA content. TP3, which has a peak absorbance at 642 nm and emission at 661 nm, has been tested on peripheral blood lymphocytes (PBL) and keratinocytes in a two-laser system. As an example, we present a three-color flow cytometric application utilizing TP3 in combination with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) conjugated to monoclonal antibodies in this paper. A subequilibrium concentration of 1 microM TP3, most preferably used in combination with RNase treatment, proved to be a powerful alternative for DNA amount determination. In human- and mouse-Balb/MK-keratinocyte populations with different S-phase fractions, PI and TP3 showed a good correlation. Finally, in the triple labelling experiment we clearly demonstrated that TP3 is readily applied to the analysis of binding of two antibodies and relative DNA content simultaneously.