Open AccessImproved single laser measurement of two cellular antigens and DNA‐ploidy by the combined use of propidium iodide and TO‐PRO‐3 iodide
Author(s) -
Corver Willem E.,
Fleuren Gert Jan,
Cornelisse Cees J.
Publication year - 1997
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(19970801)28:4<329::aid-cyto9>3.0.co;2-6
Subject(s) - propidium iodide , fluorescence , pi , microbiology and biotechnology , flow cytometry , fluorescein isothiocyanate , keratin , biophysics , materials science , biology , optics , biochemistry , physics , apoptosis , programmed cell death , paleontology
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 μM). The effect of adding a range of TP3 concentrations (0.001 to 16 μM) 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 μM in the presence of PI (100 μM). 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. Cytometry 28:329–336, 1997. © 1997 Wiley‐Liss, Inc.