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, TOPRO-3 iodide (TP3), which is best ex cited by an HeNe laser (633 nm), has been compared with propidium iodi de (PI) for measuring relative DNA content. TP3, which has a peak abso rbance at 642 nn and emission at 661 nm, has been tested on peripheral blood lymphocytes (PBL) and keratinocytes in a two-laser system. As a n example, we present a three-color flow cytometric application utiliz ing TP3 in combination with fluorescein-isothiocyanate (FITC) and phyc oerythrin (PE) conjugated to monoclonal antibodies in this paper. A su bequilibrium concentration of 1 mu M TP3, most preferably used in comb ination with RNase treatment, proved to be a powerful alternative for DNA amount determination. In human- and mouse-Balb/MK-keratinocyte pop ulations with different S-phase fractions, PI and TP3 showed a good co rrelation. Finally, in the triple labelling experiment we clearly demo nstrated that TP3 is readily applied to the analysis of binding of two antibodies and relative DNA content simultaneously. (C) 1994 Wiley-Li ss, Inc.