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 μM 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. © 1994 Wiley‐Liss, Inc.