Wessels J M, Nüsse M
GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Biophysikalische Strahlenforschung, Oberschleissheim, Germany.
Cytometry. 1995 Mar 1;19(3):201-8. doi: 10.1002/cyto.990190303.
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 and DPH were comparable to the results obtained with the combination of EB and HO.
本文介绍了一种新的染色方法，用于在细胞培养物和人淋巴细胞中通过流式细胞术测量微核（MN），该方法除了进行DNA染色外，还使用膜特异性荧光染料。研究了几种荧光膜染料和DNA染料的组合，以便在细胞核和微核悬浮液中更好地将微核与碎片区分开来。对于膜染色，亲脂性染料2-羟乙基-7,12,17-三（甲氧基乙基）卟啉（HEPn）和1,6-二苯基-1,3,5-己三烯（DPH）与溴化乙锭（EB）、硫酸原黄素（PF）和Hoechst 33258（HO）联合使用。由于它们的光谱特性，HO或EB与HEPn联合使用时，在区分微核与碎片方面不如HEPn与PF联合使用合适。然而，当HEPn与PF联合使用时，在低荧光强度下发现了额外的噪声，这可能是由于溶液中游离的荧光染料分子所致。使用DPH和EB的组合可实现膜和DNA的最佳同时染色。用这种新的染色技术研究了UV-B照射对中国仓鼠和小鼠NIH-3T3细胞中微核的诱导作用。UV-B照射（280 - 360 nm）在两种细胞系中均诱导了微核。发现中国仓鼠细胞对这些波长更敏感。波长高于360 nm的照射在两种细胞系中均未诱导微核。使用EB和DPH组合从人淋巴细胞获得的结果与使用EB和HO组合获得的结果相当。
