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