Technical note
An improved method for isolating intraepithelial lymphocytes (IELs) from the murine small intestine with consistently high purity

https://doi.org/10.1016/j.jim.2005.10.008Get rights and content

Abstract

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.

Introduction

Because intestinal intraepithelial lymphocytes (IELs) are intimately associated with epithelial cells and are dispersed throughout the epithelial layer, procedures designed to obtain IELs without epithelial cell contamination have presented special challenges for immunologists. Over the years, a number of strategies have been developed for obtaining enriched preparations of intestinal IELs from human, mouse, and rat small and large bowel (Chiba et al., 1981, Mosley and Klein, 1992, Lundquist et al., 1992, Ebert and Roberts, 1995, Santhi and Ramanadham, 1995, Kearsey and Stadnyk, 1996, Todd et al., 1999, Leon and Roy, 2004). Although it is possible to use high-speed cell sorting or magnetic-activated cell sorting for IEL purification (Shires et al., 2001, Leon and Roy, 2004, Wang et al., 2004), those techniques may be impractical on a daily basis and may yield low overall cell recoveries. Thus, most techniques utilize gradients involving synthetic polymers that separate cells based on properties of buoyant density. A common approach used for this involves the centrifugation of cells through a low density (30–45%) Percoll suspension layered on top of a high density (60–70%) Percoll suspension (Lundquist et al., 1992, Mosley and Klein, 1992, Ebert and Roberts, 1995, Kearsey and Stadnyk, 1996, Leon and Roy, 2004). Levels of purity using this range widely from 50% to 75%. In the present paper, we describe a simple modification of a standard IEL isolation technique that involves the use of two sequential Percoll centrifugation steps. This technique, while adding little additional time to the extraction procedure, consistently results in > 25% increase in IEL purity compared to single gradient purification procedures, and results in an overall purity of IELs in the range of 80–85%.

Section snippets

Mice

Adult female C57BL/6 mice, 6–8 weeks of age, were purchased from Harlan Spargue–Dawley, Indianapolis, IN. Animals were used according to protocols approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at Houston.

Media and reagents

Ca2+, Mg2+ free PBS, RPMI-1640, FBS, penicillin–streptomycin, l-glutamine, β-mercaptoethanol, Percoll (1.130 ± 0.005 g/ml), and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Sigma-Aldrich, St. Louis, MO. RPMI-1640

The use of a second Percoll gradient significantly and consistently improves the purity of IELs

Experiments were done to compare IEL purity in cell preparations after the first and second Percoll gradients. From a large series of IEL isolates (N = 14), there was a significant increase in the purity of IELs in the cell preparation after the second gradient (82.2%) compared to the purity after the first gradient (56.3%), thus representing an increase in purity of 25.9% overall (Fig. 1A). Those findings confirmed the advantage of using the two-gradient system for improving the purity of IELs

Acknowledgement

This work was supported by NIH grant DK35566.

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