Flow cytometric detection of micronuclei by combined staining of DNA and membranes
- Institut fuer Biophysikalische Strahlenforschung, Oberschleibheim (Germany)
A new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membrane-specific fluorescent dyes in addition to DNA staining. Several combinations of fluorescent membrane and DNA dyes were studied for a better discrimination of MN from debris in a suspension of nuclei and micronuclei. For staining of membranes, the lipophilic dyes 2-hydroxyethyl-7,12,17-tris(methoxyethyl)porphycene (HEPn) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were used in combination with ethidium bromide (EB), proflavine (PF), and Hoechst 33258 (HO). Due to their spectral properties, HO or EB combined with HEPn were not as suitable for the discrimination of MN from debris as was HEPn in combination with PF. With HEPn in combination with PF, however, additional noise was found at low fluorescence intensities, probably due to free fluorescent dye molecules in the solution. The optimal simultaneous staining of membranes and DNA was obtained using a combination of DPH and EB. The induction of MN in Chinese hamster and mouse NIH-3T3 cells by UV-B illumination was studied with this new staining technique. UV-B illumination (280-360 nm) induced MN in both cell lines. Chinese hamster cells were found to be more sensitive to these wavelengths. Illumination with wavelengths above 360 nm did not induce MN in either cell line. The results obtained from human lymphocytes using the combination of EB or DPH were comparable to the results obtained with the combination of EB and HO. 23 refs., 7 figs.
- Sponsoring Organization:
- USDOE
- OSTI ID:
- 456799
- Journal Information:
- Cytometry, Vol. 19, Issue 3; Other Information: PBD: 1 Mar 1995
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
APPLIED STUDIES
55 BIOLOGY AND MEDICINE
BASIC STUDIES
CELL FLOW SYSTEMS
AUTOMATION
EVALUATION
CELL NUCLEI
GENETIC RADIATION EFFECTS
MUTATION FREQUENCY
GENETIC EFFECTS
MUTATIONS
DYES
ABSORPTION
CHEMICAL PROPERTIES
SORPTIVE PROPERTIES
DETECTION
DNA
FLUORESCENCE
CELL MEMBRANES
IONIZING RADIATIONS
LYMPHOCYTES