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Isolation and analysis of group 2 innate lymphoid cells in mice

Abstract

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.

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Figure 1: Stepwise procedure for collection of BALF (steps 1–17, Box 1) and lung ILC2s (steps 1–4, Box 2).
Figure 2: Stepwise procedure for the isolation of LP ILC2s from the small intestine (steps 1–20, Box 3).
Figure 3: Stepwise procedure for the dissection of the mesentery (Steps 1–11 of the main PROCEDURE).
Figure 4: Representative gating strategy for the identification of ILC2s in various tissues.
Figure 5: Digestion of the mesentery and recovery of the stromal vascular fraction from a single mouse (Step 12A(i–vii) of the main PROCEDURE).
Figure 6: Digestion of mesenteric adipose pooled from 20 mice in a gentleMACS C tube (Step 12B(i–viii) of the main PROCEDURE).

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Acknowledgements

We thank M. Mochitzuki and N. Takeno for technical assistance. We also thank S. Wada, C. Yamamoto and T. Shitamichi for taking care of mice. Thanks are also due to J. Furusawa, T. Sasaki and S. Koga for helpful advice and discussions. This work was supported by PRESTO to K.M. from the Japan Science and Technology Agency; a Grant-in Aid for Scientific Research (B) (26293110) and a Grant-in-Aid for Challenging Exploratory Research (24659373) to K.M.; a Grant-in-Aid for Scientific Research (S) (22229004) and a Grant-in-Aid for Challenging Exploratory Research (25670235) to S.K. from the Japan Society for the Promotion of Science; a Grant-in-Aid for Scientific Research on Innovative Areas (23118526 and 15H01166) to K.M. from the Ministry of Education, Culture, Sports, Science and Technology, Japan; and grants from The Nakajima Foundation and Mochida Memorial Foundation for Medical and Pharmaceutical Research to K.M.

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K.M. and H.K. designed the protocols; K.M. and K.N.E. analyzed the results; and K.M., K.N.E. and S.K. wrote the manuscript.

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Correspondence to Kazuyo Moro.

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Competing interests

S.K. is a consultant for Medical and Biological Laboratories Co., Ltd. All other authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Stepwise procedure for opening the mouse abdominal cavity.

(a) Make a midline incision on the surface of the abdominal cavity. (b) Using hands, pull the skin to expose the abdominal cavity. Make a vertical cut through the skin. (c-d) Make 4 diagonal cuts along the abdominal cavity to expose the intestines. The blue arrows indicate the direction of the incisions.

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Moro, K., Ealey, K., Kabata, H. et al. Isolation and analysis of group 2 innate lymphoid cells in mice. Nat Protoc 10, 792–806 (2015). https://doi.org/10.1038/nprot.2015.047

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