Isolation of human small bowel intraepithelial lymphocytes by annexin V-coated magnetic beads.
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-V-coated magnetic beads, originally designed for the removal of dead cells from cell cultures, to obtain >95% 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.
León
Francisco
F
Department of Immunology, Hospital Ramón y Cajal, Madrid, Spain. fleon@niaid.nih.gov
Roy
Garbiñe
G
D017304:Annexin A5; D002460:Cell Line; D003601:Cytotoxicity Tests, Immunologic; D004386:Duodenum / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007082:Ileum / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D018189:Immunomagnetic Separation / Q000379:methods*; D007413:Intestinal Mucosa / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D010641:Phenotype; D010718:Phosphatidylserines / Q000378:metabolism; D013194:Staining and Labeling
D017304:Annexin A5; D010718:Phosphatidylserines