Since their discovery, innate lymphoid cells (ILCs) have gradually been gaining greater relevance in the field of immunology due to their multiple functions in the innate immune response. They can mainly be found in mucosal and barrier organs like skin, gut, and lungs, and have been classified into five main types (NKs, ILC1s, ILC2s, ILC3s, and Lti cells) according to their function and development. They all play major roles in functions such as tissue homeostasis, early pathogen defense, regulation of inflammation, or tissue remodeling. ILCs are mostly tissue-resident cells tightly bound to the tissue structure, a fact that requires long and complex protocols that do not always provide sufficient yield for analysis. This suggests the need for optimized approaches aimed at ensuring that enriched and viable ILC samples are obtained, in order to furnish quality results. Herein a detailed protocol is established for obtaining a single-cell suspension highly enriched in lymphoid cells from mouse gut in order to identify the different subsets of ILCs by means of flow cytometry. The cell marker panel and flow cytometry gating strategies for identification and quantification of all the different ILC populations are provided for simultaneous analysis. Moreover, the protocol described includes a procedure for studying the different cytokines produced by ILC3s involved in maintaining the integrity of the gut barrier and defending against extracellular pathogens. As a result, herein an efficient method is presented for studying mouse ILCs within the lamina propria of the small intestine and colon; this can constitute a useful tool for future investigations in the field.

自发现以来，先天淋巴样细胞（ILC）由于其在先天免疫应答中的多种功能，已逐渐在免疫学领域变得越来越重要。它们主要存在于皮肤，肠道和肺等粘膜和屏障器官中，并根据其功能和发育分为五种主要类型（NK，ILC1，ILC2，ILC3和Lti细胞）。它们均在诸如组织稳态，早期病原体防御，炎症调节或组织重塑等功能中起主要作用。ILC主要是与组织结构紧密结合的驻留组织的细胞，这一事实需要漫长而复杂的协议，而这些协议并不总是为分析提供足够的产量。这表明需要针对旨在确保获得丰富且可行的ILC样品的优化方法，为了提供高质量的结果。在本文中，建立了用于从小鼠肠中获得高度富含淋巴细胞的单细胞悬液的详细协议，以便通过流式细胞术鉴定ILC的不同亚群。提供了用于所有不同ILC群体鉴定和定量的细胞标志物组和流式细胞术门控策略，用于同时分析。此外，所描述的方案包括研究由ILC3产生的不同细胞因子的过程，这些细胞因子参与维持肠道屏障的完整性和防御细胞外病原体。结果，本文提出了一种用于研究小肠和结肠固有层内的小鼠ILC的有效方法。这可以构成将来对该领域进行调查的有用工具。