Quantitative isolation of mouse and human intestinal intraepithelial lymphocytes by elutriation centrifugation.
Intestinal intraepithelial lymphocytes (IEL) are specialized subsets of T cells with distinct functional capacities. While some IEL subsets are circulating, others such as CD8alphaalpha TCRalphabeta IEL are believed to represent non-circulating resident T cell subsets [Sim, G.K., Intraepithelial lymphocytes and the immune system. Adv. Immunol., 1995. 58: 297-343.]. Current methods to obtain enriched preparations of intraepithelial lymphocytes are mostly based on Percoll density gradient or magnetic bead-based technologies [Lundqvist, C., et al., Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine. J. Immunol. Methods, 1992. 152(2): 253-263.]. However, these techniques are hampered by a generally low yield of isolated cells, and potential artifacts due to the interference of the isolation procedure with subsequent functional assays, in particular, when antibodies against cell surface markers are required. Here we describe a new method for obtaining relatively pure populations of intestinal IEL (55-75%) at a high yield (>85%) by elutriation centrifugation. This technique is equally suited for the isolation and enrichment of intraepithelial lymphocytes of both mouse and human origin. Time requirements for fractionating cell suspensions by elutriation centrifugation are comparable to Percoll-, or MACS-based isolation procedures. Hence, the substantially higher yield and the consistent robust enrichment for intraepithelial lymphocytes, together with the gentle treatment of the cells during elutriation that does not interfere with subsequent functional assays, are important aspects that are in favor of using this elegant technology to obtain unmanipulated, unbiased populations of intestinal intraepithelial lymphocytes, and, if desired, also of pure epithelial cells.
Binda
Elisa
E
Institute of Pathology, University of Bern, Bern, Switzerland.
Erhart
Dominik
D
Schenk
Mirjam
M
Zufferey
Christel
C
Renzulli
Pietro
P
Mueller
Christoph
C
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002498:Centrifugation / Q000379:methods*; D006801:Humans; D007413:Intestinal Mucosa / Q000276:immunology*; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL