Isolation of human small bowel intraepithelial lymphocytes by Annexin V-coated magnetic beads

Abstract

Human intestinal intraepithelial lymphocytes (IEL) are important effector cells of the mucosal immune system and their study is hampered by the difficulty of their isolation. The molecular study of enriched samples of IEL is mandatory in the diagnosis of enteropathy-associated T-cell lymphoma and refractory celiac sprue. In order to isolate human small bowel IEL, we took advantage of the stress that intestinal epithelial cells (IEC) suffer during the conventional initial steps of IEL isolation, which induces their apoptosis but not that of IEL. After cell individualization by dithiothreitol and ethylenediamine tetraacetic acid, two-thirds of human IEC can be stained with Annexin-V due to their surface exposure of phosphatidyl serine, a sign of apoptosis. This percentage increases to 95% after performing a density gradient to enrich for IEL. This allows for the use of Annexin-Vcoated magnetic beads, originally designed for the removal of dead cells from cell cultures, to obtain 495% pure, 99% viable and untouched IEL after two rounds of depletion. This simple procedure has proven useful for the isolation of human IEL for functional and molecular studies and can conceivably facilitate the diagnosis of intestinal lymphoid malignancies that rely upon the study of pure IEL preparations.

Isolation and Characterization of Intestinal Epithelial Cells from Normal and SIV-Infected Rhesus Macaques

“…The dead cells were excluded by live/dead staining for all ECs and CD45 leukocytes analysis. Both DTT (reducing agent) and EDTA (cation chelant) solution has been used for the isolation of intestinal ECs [21], [22]. However, the purity of EC from each isolation technique has not been clearly defined, where the characterization of ECs was based on low to moderately sensitive immunohistochemistry techniques.…”

Under-water superoleophobicity of fish scales

“…We have followed an established protocol 16 17 for removing the mucus layer from the fish scales and we have further performed Fourier transform infrared spectroscopy (FTIR) to distinctly demarcate the presence and absence of mucus layer from the fish scales. All experimental protocols were approved by Risk Management Services, University of Alberta and methods were carried out in accordance with Biosafety Officer, Environment, Health and Safety Risk Management Services, University of Alberta.…”

Isolation and cytokine analysis of lamina propria lymphocytes from mucosal biopsies of the human colon

“…We have optimized analyses of intestinal lymphocytes from pinch biopsies taken from CD and UC patients using histo-pathological data from paired mucosal biopsies to inform us of the inflammatory state of the biopsied site[9]. Many previous methods for cellular isolation from biopsies have first used dithiothreitol (DTT) and/or ethylene diamine-tetraacetic (EDTA) to strip intraepithethial lymphocytes (IELs) from the mucosa [20]. Our method simply digests biopsies with collagenase and DNase and the subsequent Percoll step separates out lymphocytes from the remaining epithelial cells and debris.…”