Isolation and analysis of group 2 innate lymphoid cells in mice.
Recent studies have identified distinct subsets of innate lymphocytes, collectively called innate lymphoid cells (ILCs), which lack antigen receptor expression but produce various effector cytokines. Group 2 ILCs (ILC2s) respond to epithelial cell-derived cytokines such as interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP), produce large amounts of type 2 cytokines, and have a key role in anti-helminth innate immunity and in the pathophysiology of allergic inflammation. The reported phenotypic characteristics of mouse ILC2s vary, depending on the tissue source and preparation method. This protocol describes improved methods for tissue-specific isolation and analysis of mouse ILC2s of high purity and yield from fat tissue, lung, bronchoalveolar lavage fluid (BALF) and small intestine. These improved methods are the result of our thorough investigation of enzymes used for tissue digestion, methods for the elimination of undesired cells, and a combination of antibodies for the detection and isolation of ILC2s. In addition, this new protocol now enables the isolation of ILC2s of high yield, even from inflamed tissues. Depending on the tissue being analyzed, it takes ∼2-4 h for isolation and flow cytometric analysis of ILC2s from the various tissues of a single mouse and ∼4-8 h to sort purified ILC2s from pooled tissues of multiple mice.
1] Laboratory for Immune Cell Systems, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Japan. [2] Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Tokyo, Japan. [3] Division of Immunobiology, Department of Medical Life Science, Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan.
Kazuyo
K
Moro
Laboratory for Immune Cell Systems, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Japan.
Kafi N
KN
Ealey
1] Laboratory for Immune Cell Systems, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Japan. [2] Division of Pulmonary Medicine, Department of Medicine, Keio University School of Medicine, Tokyo, Japan.
Hiroki
H
Kabata
1] Laboratory for Immune Cell Systems, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Japan. [2] Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan.
Shigeo
S
Koyasu
D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology / Q000276:immunology; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D007113:Immunity, Innate; D007421:Intestine, Small / Q000166:cytology / Q000276:immunology; D008168:Lung / Q000166:cytology / Q000276:immunology; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008808:Mice, Inbred CBA