Isolation of murine small intestinal intraepithelial γδt cells.
The study of intestinal intraepithelial γδ lymphocytes has been hindered by the difficulty of isolating a population of pure cells representative of their in vivo phenotype and function. Here, we describe a procedure for the purification of small intestinal intraepithelial γδ T lymphocytes in BALB/c mice, including extraction and purification of intraepithelial lymphocytes (IELs) followed by separation of γδ T cells from IELs by magnetic activated cell sorting (MACS). The small intestinal IEL isolation protocol reported here is a less time-consuming with high purity and high yields. One-step Percoll purification of IELs using a 40%-70% discontinuous Percoll gradient is less laborious yet sufficient to get exact profiles during phenotypic analysis of cell subsets. IELs obtained after two-step Percoll centrifugation using 30% Percoll, followed by a 40%-70% discontinuous Percoll gradient, are fit to purify γδ T cells by MACS. Small intestinal IELs produce much more IFN-γ and TGF-β1 than sorted γδ T cells upon stimulation with plate-bound anti-CD3ε mAb. γδ T cells secret moderate amounts of TGF-β1 and minimal amounts of IFN-γ. Purification of γδ T cells is helpful in investigating the phenotype and function of intestinal mucosal γδ T cells.
Ye
Yuefang
Y
Department of Gastroenterology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, PR China.
Yue
Min
M
Jin
Xi
X
Chen
Shaohua
S
Li
Youming
Y
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D005434:Flow Cytometry; D018189:Immunomagnetic Separation; D007413:Intestinal Mucosa / Q000473:pathology*; D007421:Intestine, Small / Q000276:immunology / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000473:pathology
D016692:Receptors, Antigen, T-Cell, gamma-delta