IMPROVED SINGLE LASER MEASUREMENT OF 2 CELLULAR ANTIGENS AND DNA-PLOIDY BY THE COMBINED USE OF PROPIDIUM-IODIDE AND TO-PRO-3-IODIDE

Recently, Frey (Cytometry 17:310-318, 1994) demonstrated that TO-PRO-3 iodide (TPS) cain be excited indirectly by a 488 nm laser Line throug h energy transfer by propidium iodide (PI). In the present study, we i nvestigated whether PI-TPS energy transfer can help to overcome spectr al cross talk problems associated with the combined use of fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), and PI. Mixtures of kera tin 8/18 FITC labeled, keratin 8/18-PE-labeled, and unlabeled MCF-7 br east carcinoma cells were prepared and stained for DNA with PI (100 mu M). The effect of adding a range of TPS concentrations (0.001 to 16 m u M) to these mixtures was evaluated. The combined use of PI and TP3 w as further evaluated using mixtures of unlabeled and p53 FITC-labeled COV362.c14 ovarian cancer cells and mixtures of unlabeled and p53 FITC -labeled COV362.c14 cells and peripheral blood lymphocytes (PBL), addi tionally stained for keratin 8/18 (PE). Finally, a human ovarian ascit es tumor specimen was triple-stained for keratin 8/18 (PE), vimentin ( FITC) and DNA or keratin 8/18 (PE), PCNA (FITC) and DNA. Addition of T P3 allowed complete correction for spectral cross talk of PE/PI into t he green fluorescence detector (FL1). Only minimal (FL1-%FL2) compensa tion was required at a TP3 concentration of 2.0 mu M in the presence o f PI (100 mu M). The PI spectral cross talk into the orange fluorescen ce detector (FL2) was reduced by about 50% using the same photomultipl ier (PMT) settings. Although addition of TPS reduced the signal-to-bac kground ratio by about 30%, the advantage gained through full compensa tion for spectral cross talk resulted in an improved discrimination of p53-positive and -negative subpopulations in a mixture of human PBL a nd COV362.c14 cells. Furthermore, vimentin-negative and PCNA-negative cells mere better resolved in a human DNA-aneuploid ovarian ascites tu mor after staining the DNA with PI/TP3, rather than with PI alone. We conclude that the addition of TP3 to PI improves the combined measurem ent by single-laser flow cytometry of DNA-ploidy and antigen expressio n in heterogenous clinical samples. (C) 1997 Wiley-Liss, Inc.