Tatcheff Michaela, Carvalho Christine, Willar Jonas, Nendel Elvedina, Krammer Susanne, Chiriac Mircea T, Zhou Shuting, Geppert Carol I, Finotto Susetta
Department of Molecular Pneumology, Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg, Universitätsklinikum Erlangen, Erlangen, Germany.
These two authors contributed equally to this work and thus share the first authorship.
Curr Protoc. 2025 Jan;5(1):e70087. doi: 10.1002/cpz1.70087.
Understanding the dynamic pathophysiology of diseases in the lung, such as asthma and chronic asthma, chronic obstructive pulmonary disease, and lung cancer, is crucial for the treatment, analysis, and outcome of these diseases. Unlike other traditional models, we suggest a protocol that is sustainable and reproducible and offers different analysis methods while maintaining in vivo lung architecture and immune dynamics. This protocol allows one to study the pathophysiological changes, including changes to the immune cells, cytokines, and mediators, in 30 precision-cut lung slices from a single murine lung. To accomplish this, the murine lung is infused with 2.5% low-melting-point agarose and is precision-cut-sliced. Our method also supports cell culture in refined medium and stimulation with clinically relevant stimuli, which helps to clarify the mechanisms of the disease. Evaluation of the samples and their supernatant includes multiplex assays, ELISA, histology, and immunohistochemistry. Additional sections are used to extract RNA for quantitative real-time PCR and RNA sequencing and/or other selected analysis, like flow cytometry. Using this method, we obtained murine lung slices that preserve the pathophysiology of the disease and allow a comprehensive analysis unlike other already-existing protocols. By retaining the dynamic immune mechanisms, we are able to see the histological damage caused by each disease. The results of this protocol can be used to improve our understanding and therapy options. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Obtaining precision-cut lung slices (PCLSs) from the murine lung Support Protocol 1: LL/2 cell treatment with G-418 solution Support Protocol 2: Murine model of lung adenocarcinoma and in vivo imaging Support Protocol 3: H&E staining of PCLS sections Support Protocol 4: Measuring cytokines by ELISA Support Protocol 5: Measuring cytotoxic activity in PCLS conditioned medium Support Protocol 6: Analysis of RNA from PCLSs by real-time PCR Support Protocol 7: Flow cytometry analysis of cells isolated from PCLSs.
了解肺部疾病（如哮喘、慢性哮喘、慢性阻塞性肺疾病和肺癌）的动态病理生理学对于这些疾病的治疗、分析和预后至关重要。与其他传统模型不同，我们提出了一种可持续且可重复的方案，该方案在保持体内肺结构和免疫动态的同时提供不同的分析方法。该方案允许研究来自单个小鼠肺的30个精密切割肺切片中的病理生理变化，包括免疫细胞、细胞因子和介质的变化。为此，向小鼠肺中注入2.5%的低熔点琼脂糖并进行精密切割切片。我们的方法还支持在精制培养基中进行细胞培养并用临床相关刺激物进行刺激，这有助于阐明疾病机制。对样品及其上清液的评估包括多重分析、酶联免疫吸附测定、组织学和免疫组织化学。额外的切片用于提取RNA进行定量实时聚合酶链反应和RNA测序和/或其他选定的分析，如流式细胞术。使用这种方法，我们获得了保留疾病病理生理学的小鼠肺切片，并允许进行与其他现有方案不同的全面分析。通过保留动态免疫机制，我们能够看到每种疾病造成的组织学损伤。该方案的结果可用于增进我们的理解和治疗选择。© 2025作者。由威利期刊有限责任公司出版的《当前方案》。基本方案：从小鼠肺中获取精密切割肺切片（PCLSs）支持方案1：用G-418溶液处理LL/2细胞支持方案2：肺腺癌小鼠模型和体内成像支持方案3：PCLS切片的苏木精-伊红染色支持方案4：通过酶联免疫吸附测定测量细胞因子支持方案5：测量PCLS条件培养基中的细胞毒性活性支持方案6：通过实时聚合酶链反应分析PCLSs中的RNA支持方案7：对从PCLSs中分离的细胞进行流式细胞术分析。
