Abstract | BACKGROUND: Multiparameter DNA flow cytometry using a one- laser bench-top flow cytometer has been restricted to three different colors. The two laser FACSCalibur has recently been introduced, allowing four-color analysis. Therefore, we optimized and extended our three-color method (Corver et al., 1994, Corver et al. 1996) to a four-color analysis of phenotypic intra- tumor heterogeneity using a bench-top flow cytometer. METHODS: RESULTS: Overall, PI produced better CVs than did TP3. The optimal concentration of PI was 50-100 microM for all cells tested. Average CVs were 1.76% (PBL), 3.16% (HeLa), and 2.50% (SiHa). Optimal TP3 concentrations were 0.25-2.0 microM. Average CVs were 2. 58% (PBL), 5.16% (HeLa), and 3.96% (SiHa). Inter- or intra- DNA stem line heterogeneity of Ep-CAM expression was observed in the keratin-positive fractions. Vimentin-positive, keratin-negative cells were restricted to the DNA diploid fraction. CONCLUSIONS: PI is a superior DNA stain to TP3 when using intact normal PBL and human cancer cells. Four-color high-resolution multiparameter DNA flow cytometry allows the identification of intratumor subpopulations using PI as DNA stain and FITC, RPE, and APC as reporter molecules. The FACSCalibur bench-top flow cytometer can be used for this purpose, allowing the application of this technique in clinical laboratories.
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Authors | W E Corver, L A Koopman, J van der Aa, M Regensburg, G J Fleuren, C J Cornelisse |
Journal | Cytometry
(Cytometry)
Vol. 39
Issue 2
Pg. 96-107
(Feb 01 2000)
ISSN: 0196-4763 [Print] United States |
PMID | 10679727
(Publication Type: Journal Article)
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Copyright | Copyright 2000 Wiley-Liss, Inc. |
Chemical References |
- Antibodies, Monoclonal
- Antigens, Neoplasm
- Carbocyanines
- Cell Adhesion Molecules
- Epithelial Cell Adhesion Molecule
- Fluorescent Dyes
- TO-PRO-3
- allophycocyanin
- Phycocyanin
- Propidium
- Keratins
- DNA
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Topics |
- Antibodies, Monoclonal
(immunology)
- Antigens, Neoplasm
(analysis, immunology)
- Carbocyanines
- Cell Adhesion Molecules
(analysis, immunology)
- DNA
(analysis)
- Epithelial Cell Adhesion Molecule
- Female
- Flow Cytometry
(instrumentation, methods)
- Fluorescent Antibody Technique
- Fluorescent Dyes
- Humans
- Keratins
(immunology)
- Lymphocytes
- Phenotype
- Phycocyanin
- Propidium
- Tumor Cells, Cultured
- Uterine Cervical Neoplasms
(genetics)
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