Swank-Hill P, Needham L K, Schnaar R L
Anal Biochem. 1987 May 15;163(1):27-35. doi: 10.1016/0003-2697(87)90088-1.
Cell surface carbohydrates and complementary carbohydrate receptors may mediate cell-cell recognition and adhesion. We report a method which detects carbohydrate-specific adhesion of intact eukaryotic cells directly to glycosphingolipids separated on thin-layer chromatography plates. Various glycosphingolipids were chromatographed on high-performance silica gel thin-layer chromatography plates, and the plates were coated with a thin film of poly(isobutyl methacrylate) and mounted in a specially designed plexiglass chamber. Metabolically radiolabeled cells were added to the chamber, which was then sealed and gently centrifuged to bring the cells into contact with the surface of the TLC plate. After incubation to allow adhesion to occur, the chamber was inverted and centrifuged to remove nonadherent cells from the plate surface. The plate was removed from the chamber, the adherent cells were fixed in place with glutaraldehyde, and the plate was dried and subjected to autoradiography. Chicken hepatocytes, which have a cell surface receptor for N-acetylglucosamine, adhered only to those areas of the plate to which appropriate glycosphingolipids (having that terminal sugar) had migrated. Cell adhesion was blocked by soluble N-acetylglucosamine (but not by other sugars) and was readily detectable using a variety of developing solvents. Cell adhesion to as little as 8 pmol of the appropriate lipid was readily detected. This method can be used to test glycosphingolipids as cell surface recognition markers for a variety of cell types.
细胞表面碳水化合物和互补的碳水化合物受体可能介导细胞间的识别与黏附。我们报道了一种直接检测完整真核细胞与在薄层层析板上分离的糖鞘脂之间碳水化合物特异性黏附的方法。将各种糖鞘脂在高效硅胶薄层层析板上进行层析，然后在这些板上涂覆一层聚（甲基丙烯酸异丁酯）薄膜，并安装在一个特别设计的有机玻璃腔室中。将经代谢放射性标记的细胞加入腔室，密封后轻轻离心以使细胞与薄层层析板表面接触。孵育以使黏附发生后，将腔室倒置并离心以从板表面去除未黏附的细胞。将板从腔室中取出，用戊二醛固定黏附的细胞，然后将板干燥并进行放射自显影。鸡肝细胞具有N - 乙酰葡糖胺的细胞表面受体，它仅黏附于板上合适的糖鞘脂（具有该末端糖）迁移到的那些区域。细胞黏附可被可溶性N - 乙酰葡糖胺（但不被其他糖类）阻断，并且使用多种展开溶剂很容易检测到。很容易检测到细胞与低至8皮摩尔合适脂质的黏附。该方法可用于测试糖鞘脂作为多种细胞类型的细胞表面识别标记。
