HOECHST 33342 DYE UPTAKE AS A PROBE OF MEMBRANE-PERMEABILITY CHANGES IN MAMMALIAN-CELLS
HOECHST 33342 DYE UPTAKE AS A PROBE OF MEMBRANE-PERMEABILITY CHANGES IN MAMMALIAN-CELLS
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DOI：
10.1073/pnas.78.1.363
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
MILLER, RG
中科院分区：
文献类型：
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作者：
LALANDE, ME;LING, V;MILLER, RG
流式细胞术分析DNA特异的荧光探针Hoechst 33342(HO342)的摄取提供了一种简单而快速的方法来测量哺乳动物细胞的膜转运速率，并识别不同的细胞亚群的膜转运速率。在中国仓鼠卵巢野生型细胞系及其衍生的3个秋水仙碱耐药系中，对HO342染料的摄取速率随着对秋水仙碱的逐步抗性的增加而降低。秋水仙素敏感的回变细胞系显示染料摄取率增加。这些菌株的耐药性以前被证明与转运率的变化有关。在与秋水仙素相似的方式下，HO342染料的吸收速率表现为非饱和动力学。代谢抑制剂KCN对HO342染料摄取的影响，在有无葡萄糖的情况下，与先前观察到的秋水仙碱摄取类似。在适当的条件下用HO342对小鼠脾细胞进行染色，可以看到两种淋巴细胞的HO342荧光强度不同，这种差异与DNA含量无关。这两个亚群在秋水仙碱和HO342的摄取方面显示出相同的相对差异。HO342染料似乎以与秋水仙素相同的机制进入哺乳动物细胞，即通过非介导性扩散，并可用作细胞质膜通透性的探针。讨论了该染料在耐药性研究、活化T细胞检测和淋巴细胞亚群识别方面的潜在应用。
Flow cytometric analysis of the uptake of the DNA-specific and fluorescent probe Hoechst 33342 (HO342) offers a simple and rapid method for measuring membrane transport rates in mammalian cells, and of identifying cellular subpopulations that differ in their membrane transport rates. In a Chinese hamster ovary wild-type cell line and in 3 colchicine-resistant lines derived from it, the rate of uptake of HO342 dye decreases as the stepwise resistance to colchicine increases. A colchicine-sensitive revertant cell line shows an increased rate of dye uptake. Drug resistance in these lines was previously shown to be related to changes in transport rate. In a manner similar to colchicine, the rate of uptake of HO342 dye shows nonsaturation kinetics. The effect of KCN, a metabolic inhibitor, on HO342 dye uptake, both in the presence and in the absence of glucose, is similar to that previously observed for colchicine uptake. When murine spleen cells are stained with HO342 under appropriate conditions, one sees 2 populations of lymphocytes differing in HO342 fluorescence intensity, a difference not related to DNA content. The 2 subpopulations show the same relative difference in both colchicine and HO342 uptake. HO342 dye appears to enter mammalian cells by the same mechanism as colchicine, i.e., by unmediated diffusion, and can be used as a probe of cytoplasmic membrane permeability. Potential applications of the dye in studies of drug resistance, detection of activated T cells, and recognition of lymphocyte subpopulations are discussed.