Benn Peter, Delach Judith
Human Genetics Laboratories, Department of Genetics and Developmental Biology, and Department of Pathology and Laboratory Medicine, University of Connecticut Health Center, Farmington, CT 06030-6140, USA.
CSH Protoc. 2008 Sep 1;2008:pdb.prot5035. doi: 10.1101/pdb.prot5035.
INTRODUCTIONPhytohaemagglutinin (PHA), a lectin derived from the red kidney bean, is a powerful mitogen for human T-cells. When PHA is added in vitro to whole blood, mitotic cells can be found after 48 h, with a peak mitotic index at ~64-72 h. The convenience of peripheral blood as a source of human cells, the abundance of mitotic cells, and the simplicity of the cell culture technique make this the most convenient approach to study human chromosomes for both clinical and research purposes. This method of chromosome preparation provides metaphase cells that can be stained by a variety of methods or used for fluorescence in situ hybridization (FISH). The most common chromosome staining techniques involve exposing fixed preparations to a protease (e.g., trypsin), followed by an appropriate semipermanent stain. The characteristic banding patterns obtained reflect both structural and functional differences in different parts of the chromosomes. The staining procedure described here provides a Giemsa banding pattern using trypsin with Wright stain (i.e., GTW banding). This procedure is reliable and, with only minor modifications, suitable for preparing chromosomes from a variety of human tissues.
引言
植物血凝素（PHA）是一种从红芸豆中提取的凝集素，是人类T细胞的强效有丝分裂原。当在体外将PHA添加到全血中时，48小时后可发现有丝分裂细胞，在约64 - 72小时达到有丝分裂指数峰值。外周血作为人类细胞来源的便利性、有丝分裂细胞的丰富性以及细胞培养技术的简单性，使其成为临床和研究目的下研究人类染色体最便捷的方法。这种染色体制备方法提供了可通过多种方法染色或用于荧光原位杂交（FISH）的中期细胞。最常见的染色体染色技术包括将固定的标本用蛋白酶（如胰蛋白酶）处理，然后进行适当的半永久性染色。获得的特征性带型反映了染色体不同部分的结构和功能差异。这里描述的染色程序使用胰蛋白酶和瑞氏染色剂提供吉姆萨带型（即GTW带型）。该程序可靠，只需进行微小修改，就适用于从各种人类组织制备染色体。
