An improved method for isolating intraepithelial lymphocytes (IELs) from the murine small intestine with consistently high purity
An improved method for isolating intraepithelial lymphocytes (IELs) from the murine small intestine with consistently high purity
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DOI：
10.1016/j.jim.2005.10.008
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发表时间：
2006-01-20
影响因子：
2.2
通讯作者：
Klein, JR
中科院分区：
文献类型：
--
作者：
Montufar-Solis, D;Klein, JR
获得肠上皮内淋巴细胞（IEL）制剂的方法，目前特殊的挑战，免疫学家由于难以回收IEL没有污染的肠细胞。虽然高纯度的制备可以使用技术，如流式细胞术或磁激活细胞分选，这些方法可能是不可行的常规基础上，并可能导致低的整体细胞回收率。因此，目前大多数程序依赖于密度梯度离心作为分离IEL和非造血细胞的手段;然而，来自这些制剂的IEL的纯度可能差异很大。在这里，我们描述了一种改进的IEL纯化技术，它使用两个连续的Percoll梯度，而不是一个梯度的纯化方案。这种改变始终导致细胞制备物中IEL纯度为80-85%。此外，它不需要额外的试剂，对回收的IEL的表型组成或细胞活力没有不利影响，并且对分离方案增加了最少的额外时间。预期该方法作为常规分离高纯度IEL的方法具有实际益处，可用于IEL功能的体内或体外研究。(c)2005 Elsevier B.V保留所有权利。
Methods for obtaining preparation of intestinal intraepithelial lymphocytes (IELs) present special challenges for immunologists due to difficulties in recovering IELs devoid of contaminating enterocytes. Although high-purity preparations can be achieved using techniques such as flow cytometric or magnetic-activated cell sorting, those methods may not be feasible on a routine basis and may result in low overall cell recoveries. Thus, most procedures today rely on density gradient centrifugation as a means of separating IEL and non-hematopoietic cells; however, the purity of IELs from those preparations can vary considerably. Here, we describe a modification of an IEL purification technique that uses two sequential Percoll gradients rather than one gradient in the purification scheme. This alteration consistently results in 80-85% IEL purity in cell preparations. Moreover, it requires no additional reagents, has no adverse effect on the phenotypic composition of recovered IELs or on the cell viability, and adds minimal additional time to the isolation protocol. It is expected that this procedure will have practical benefit as a means of isolating IELs with high purity on a routine basis that can be used for in vivo or in vitro studies of IEL function. (c) 2005 Elsevier B.V All rights reserved.