The labeling of lipoproteins for studies of cellular binding with a fluorescent lipophilic dye.
The labeling of lipoproteins for studies of cellular binding with a fluorescent lipophilic dye.
复制标题
脂蛋白标记，用于研究细胞与荧光亲脂染料的结合。
DOI：
10.1016/0003-2697(91)90306-e
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发表时间：
1991
影响因子：
2.9
通讯作者：
Sparks,CE
中科院分区：
文献类型：
--
作者：
Corsetti,JP;Weidner,CH;Cianci,J;Sparks,CE
碘化N，N-双十五烷基氨基苯乙烯基吡啶鎓是一种染料，其在脂质中的荧光强度比在水性环境中强约100倍。这一观察结果表明其作为脂蛋白荧光染色剂的潜力。这项工作报告的染色LDL与这种染料用于细胞结合的研究。染色程序简单，导致染料的稳定附着，如通过转移实验所确定的，物理性质基本上与天然LDL相同，如通过几乎相同的电泳迁移率所证明的，并且在使用流式细胞术的细胞结合研究中结果一致。与用于脂蛋白染色的其他染料（包括广泛使用的Dil（3，3 ′-双十八烷基吲哚羰花青碘化物））相比，信噪比增加，可实现更高灵敏度和精确度的测定。这通过流式细胞术测定LDL与新鲜分离的人外周血淋巴细胞（即，LDL受体未上调的细胞）。介导的LDL受体的结合证明了从这项工作和文献值的LDL受体解离常数之间的对应关系;增加在脂蛋白缺乏的培养基中培养的淋巴细胞的特异性结合，以上调LDL受体;和减少在25-羟基胆固醇的存在下培养48小时的淋巴细胞的特异性结合，以抑制LDL受体。
N,N-dipentadecylaminostyrylpyridinium iodide is a dye that is approximately 100-fold more intensely fluorescent in a lipid than aqueous environment. This observation suggests its potential as a fluorescence stain for lipoproteins. This work reports the staining of LDL with this dye for use in studies of cellular binding. The staining procedure is simple, resulting in stable attachment of the dye as determined by transfer experiments, physical properties essentially identical to native LDL as demonstrated by virtually identical electrophoretic mobility, and consistent results in studies of cellular binding using flow cytometry. Increased signal to noise ratio over other dyes used for lipoprotein staining including the widely used Dil (3,3′-dioctadecylindocarbocyanine iodide) allows determinations of greater sensitivity and precision to be made. This is demonstrated by the flow cytometric determination of the 4°C binding curve of LDL with freshly isolated human peripheral blood lymphocytes (i.e., cells not LDL receptor upregulated). Mediation of binding by the LDL receptor is demonstrated by correspondence between the LDL receptor dissociation constant derived from this work and literature values; increased specific binding in lymphocytes cultured in lipoprotein-deficient media to up-regulate the LDL receptor; and decreased specific binding in lymphocytes cultured in the presence of 25-hydroxy cholesterol for 48 h to suppress the LDL receptor.