Intact cell adhesion to glycan microarrays
Intact cell adhesion to glycan microarrays
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DOI：
10.1093/glycob/cwh022
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发表时间：
2004-02-01
期刊：
影响因子：
4.3
通讯作者：
Schnaar, RL
中科院分区：
文献类型：
--
作者：
Nimrichter, L;Gargir, A;Schnaar, RL
开发了一种快速且可重复的方法来检测和定量碳水化合物介导的细胞与载玻片上排列的聚糖的粘附。将单糖和寡糖以1.7 mm直径的斑点（200个斑点/载玻片）共价连接到载玻片上，斑点由Teflon垫圈隔开。原代鸡肝细胞组成型表达与非还原末端N-乙酰葡糖胺残基结合的C型凝集素，用荧光染料标记，并在糖基化斑点上以1.3-穆尔等分试样孵育。在孵育以允许细胞粘附后，通过将载玻片浸入磷酸盐缓冲盐水中，倒置并在密封的定制丙烯酸室中放置来去除未粘附的细胞，使得衍生化斑点上的细胞经受均匀且受控的离心分离力，同时避免空气-液体界面。离心后，将贴壁细胞固定在适当位置并通过荧光成像检测。鸡肝细胞以不同的连接和方向与非还原性末端GlcNAc残基结合，但不与非还原性末端半乳糖或N-乙酰半乳糖胺残基结合。在孵育之前添加可溶性GlcNAc（但不是Gal）将细胞粘附降低至背景水平。将该方法扩展到45-聚糖多样性阵列上的CD 4（+）人T细胞，显示了对唾液酸刘易斯x结构的特异性粘附。所描述的方法是使用各种聚糖定量选择性细胞粘附的稳健方法，并且可能有助于糖组学研究的工具库。
A rapid and reproducible method was developed to detect and quantify carbohydrate-mediated cell adhesion to glycans arrayed on glass slides. Monosaccharides and oligosaccharides were covalently attached to glass slides in 1.7-mm-diameter spots (200 spots/slide) separated by a Teflon gasket. Primary chicken hepatocytes, which constitutively express a C-type lectin that binds to nonreducing terminal N-acetylglucosamine residues, were labeled with a fluorescent dye and incubated in 1.3-muL aliquots on the glycosylated spots. After incubating to allow cell adhesion, nonadherent cells were removed by immersing the slide in phosphate buffered saline, inverting, and centrifuging in a sealed custom acrylic chamber so that cells on the derivatized spots were subjected to a uniform and controlled centrifugal detachment force while avoiding an air-liquid interface. After centrifugation, adherent cells were fixed in place and detected by fluorescent imaging. Chicken hepatocytes bound to nonreducing terminal GlcNAc residues in different linkages and orientations but not to nonreducing terminal galactose or N-acetylgalactosamine residues. Addition of soluble GlcNAc (but not Gal) prior to incubation reduced cell adhesion to background levels. Extension of the method to CD4(+) human T-cells on a 45-glycan diversity array revealed specific adhesion to the sialyl Lewis x structure. The described method is a robust approach to quantify selective cell adhesion using a wide variety of glycans and may contribute to the repertoire of tools for the study of glycomics.