McGowan P F, Hurst R E, Bass R A, Wilcox L J, Hemstreet G P, Postier R G
Department of Surgery, Oklahoma University Health Sciences Center, Oklahoma City 73190.
J Histochem Cytochem. 1988 Jul;36(7):757-62. doi: 10.1177/36.7.2454985.
We examined the biophysical characteristics of the interaction of Hoechst 33258 and 33342 dyes with normal rat colorectal cells as functions of fixation and solution composition. Classical dye-binding techniques were used to investigate the stoichiometry and binding constants with whole cells, and quantitative fluorescence image analysis was used to specifically study nuclear dye binding in intact cells. In aqueous solution, H-33258 dye bound cooperatively with intact cells, with a binding constant of between 3-4 x 10(5). In ethanolic solution, binding appeared less cooperative, although Scatchard analysis could not be used. The binding constant was slightly lower (2 x 10(5)), but the total number of cell binding sites was decreased by a factor of 5, reflecting a great decrease in cytoplasmic sites. QFIA studies identified conditions optimal for DNA quantitation under which the fluorescence signal was independent of dye or cell concentration. The proportionality between absolute nuclear fluorescence intensity and DNA content was established, and the upper limit of DNA content of normal colorectal cells was also determined.
我们研究了Hoechst 33258和33342染料与正常大鼠结肠直肠细胞相互作用的生物物理特性，这些特性是固定和溶液组成的函数。采用经典的染料结合技术研究与全细胞的化学计量和结合常数，并使用定量荧光图像分析专门研究完整细胞中的核染料结合。在水溶液中，H-33258染料与完整细胞协同结合，结合常数在3-4×10⁵之间。在乙醇溶液中，结合似乎不太协同，尽管无法使用Scatchard分析。结合常数略低（2×10⁵），但细胞结合位点的总数减少了5倍，这反映了细胞质位点的大幅减少。QFIA研究确定了DNA定量的最佳条件，在这些条件下荧光信号与染料或细胞浓度无关。建立了绝对核荧光强度与DNA含量之间的比例关系，并确定了正常结肠直肠细胞DNA含量的上限。
