Singh Ashish K, Blanco Alfonso, Sinnott Ray, Knaus Ulla G
Conway Institute, School of Medicine, University College Dublin, Dublin, Ireland.
Flow Cytometry Core, Conway Institute, University College Dublin, Dublin, Ireland.
Bio Protoc. 2025 Aug 20;15(16):e5417. doi: 10.21769/BioProtoc.5417.
Chemically induced murine colitis models are widely used to understand intestinal homeostasis and inflammatory responses during acute and chronic gut inflammation, such as inflammatory bowel disease (IBD). Resident populations of immune cells, together with those recruited during an inflammatory response, maintain intestinal immunity by mounting an effective immune response to enteropathogenic microbes while at the same time maintaining tolerance against commensals. To better understand the disease mechanism, studying different immune cell populations and their dynamic changes during infection and inflammation is essential. However, isolating healthy and viable immune populations, particularly hyperactivated neutrophils and macrophages from the inflamed gut (i.e., active disease site), is challenging as tissues are usually subjected to rigorous enzymatic digestion for an extended period. Here, we describe a method that uses a cell dissociator (Medimachine II from Syntec International) to separate intestinal tissue after short enzymatic digestion to obtain a single-cell suspension. This technique facilitates the isolation of immune cells from mouse intestinal tissues in high quantity and with superior viability in a very short time frame. This protocol delivers 80%-90% cell viability, which is 1.5 to 2-fold higher than conventional methods of isolating cells from inflamed mouse colons. The composition, phenotype, activation state, and gene expression profile of cells isolated using this protocol can be assessed by using multiple methods, including, but not limited to, flow cytometry, quantitative PCR, immunoblotting, mass spectrometry, single-cell RNA sequencing, and functional readouts such as reactive oxygen species (ROS) production. Key features • Infiltrating immune cells are key drivers of intestinal inflammation. • Isolating viable cells from the inflamed colon is slow and may alter cell activation and expression profiles. • This protocol enables faster isolation with improved cell viability. • Isolated cells can be further purified using magnetic beads or flow cytometry for downstream analysis.
化学诱导的小鼠结肠炎模型被广泛用于了解急性和慢性肠道炎症（如炎症性肠病，IBD）期间的肠道稳态和炎症反应。免疫细胞的常驻群体以及炎症反应期间募集的细胞，通过对肠道致病微生物发起有效的免疫反应，同时维持对共生菌的耐受性，来维持肠道免疫。为了更好地理解疾病机制，研究不同免疫细胞群体及其在感染和炎症期间的动态变化至关重要。然而，从发炎的肠道（即活跃疾病部位）分离健康且有活力的免疫群体，尤其是过度活化的中性粒细胞和巨噬细胞具有挑战性，因为组织通常需要长时间进行严格的酶消化。在此，我们描述一种方法，该方法使用细胞解离器（Syntec International公司的Medimachine II）在短时间酶消化后分离肠道组织，以获得单细胞悬液。这项技术有助于在极短时间内从小鼠肠道组织中大量分离免疫细胞，且细胞活力极佳。该方案可实现80%-90%的细胞活力，比从发炎的小鼠结肠中分离细胞的传统方法高出1.5至2倍。使用该方案分离的细胞的组成、表型、活化状态和基因表达谱可通过多种方法进行评估，包括但不限于流式细胞术、定量PCR、免疫印迹、质谱分析、单细胞RNA测序以及诸如活性氧（ROS）产生等功能读数。关键特性 • 浸润的免疫细胞是肠道炎症的关键驱动因素。 • 从发炎的结肠中分离活细胞速度缓慢，且可能改变细胞活化和表达谱。 • 本方案能够更快地分离细胞，同时提高细胞活力。 • 分离的细胞可使用磁珠或流式细胞术进一步纯化，用于下游分析。
