title: Improved single laser measurement of two cellular antigens and DNA-ploidy by the combined use of propidium iodide and TO-PRO-3 iodide.
abstract: Recently, Frey (Cytometry 17:310-318, 1994) demonstrated that TO-PRO-3 iodide (TP3) can be excited indirectly by a 488 nm laser line through energy transfer by propidium iodide (PI). In the present study, we investigated whether PI-TP3 energy transfer can help to overcome spectral cross talk problems associated with the combined use of fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), and PI. Mixtures of keratin 8/18 FITC-labeled, keratin 8/18-PE-labeled, and unlabeled MCF-7 breast carcinoma cells were prepared and stained for DNA with PI (100 microM). The effect of adding a range of TP3 concentrations (0.001 to 16 microM) to these mixtures was evaluated. The combined use of PI and TP3 was further evaluated using mixtures of unlabeled and p53 FITC-labeled COV362.cl4 ovarian cancer cells and mixtures of unlabeled and p53 FITC-labeled COV362.cl4 cells and peripheral blood lymphocytes (PBL), additionally stained for keratin 8/18 (PE). Finally, a human ovarian ascites 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 TP3 allowed complete correction for spectral cross talk of PE/PI into the green fluorescence detector (FL1). Only minimal (FL1 - %FL2) compensation was required at a TP3 concentration of 2.0 microM in the presence of PI (100 microM). The PI spectral cross talk into the orange fluorescence detector (FL2) was reduced by about 50% using the same photomultiplier (PMT) settings. Although addition of TP3 reduced the signal-to-background ratio by about 30%, the advantage gained through full compensation for spectral cross talk resulted in an improved discrimination of p53-positive and -negative subpopulations in a mixture of human PBL and COV362.cl4 cells. Furthermore, vimentin-negative and PCNA-negative cells were better resolved in a human DNA-aneuploid ovarian ascites tumor after staining the DNA with PI/TP3, rather than with PI alone. We conclude that the addition of TP3 to PI improves the combined measurement by single-laser flow cytometry of DNA-ploidy and antigen expression in heterogenous clinical samples.
authors.affiliation: Department of Pathology, Faculty of Medicine, Leiden University, The Netherlands. CORV@pathology.medfac.leidenuniv.nl
authors.forename: W E
authors.initials: WE
authors.lastname: Corver
authors.forename: G J
authors.initials: GJ
authors.lastname: Fleuren
authors.forename: C J
authors.initials: CJ
authors.lastname: Cornelisse
mesh_terms: D000941:Antigens / Q000032:analysis; D001201:Ascites; D002232:Carbocyanines; D004273:DNA, Neoplasm / Q000032:analysis*; D004735:Energy Transfer; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007633:Keratins / Q000032:analysis*; D007834:Lasers; D010051:Ovarian Neoplasms / Q000235:genetics / Q000276:immunology; D011003:Ploidies; D011419:Propidium; D017186:Titrimetry; D014407:Tumor Cells, Cultured
chemical_list: D000941:Antigens; D002232:Carbocyanines; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; C098830:TO-PRO-3; D011419:Propidium; D007633:Keratins