体外细胞克隆形成试验。

Clonogenic assay of cells in vitro.

作者信息

Franken Nicolaas A P, Rodermond Hans M, Stap Jan, Haveman Jaap, van Bree Chris

机构信息

Laboratory for Experimental Oncology and Radiobiology, Department of Radiotherapy, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands.

出版信息

Nat Protoc. 2006;1(5):2315-9. doi: 10.1038/nprot.2006.339.

DOI:10.1038/nprot.2006.339

PMID:17406473

Abstract

Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is defined to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo "unlimited" division. Clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. Only a fraction of seeded cells retains the capacity to produce colonies. Before or after treatment, cells are seeded out in appropriate dilutions to form colonies in 1-3 weeks. Colonies are fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using a stereomicroscope. A method for the analysis of radiation dose-survival curves is included.

摘要

克隆形成试验或集落形成试验是一种基于单个细胞生长成集落能力的体外细胞存活试验。集落定义为由至少50个细胞组成。该试验本质上测试群体中的每个细胞进行“无限”分裂的能力。克隆形成试验是确定电离辐射处理后细胞生殖死亡的首选方法，但也可用于确定其他细胞毒性剂的有效性。只有一小部分接种的细胞保留产生集落的能力。在处理之前或之后，将细胞以适当的稀释度接种，在1至3周内形成集落。用戊二醛（6.0% v/v）固定集落，用结晶紫（0.5% w/v）染色，并用体视显微镜计数。文中还包括一种分析辐射剂量-存活曲线的方法。