Analyzing Pathophysiology and Immune Cells and Their Cytokines and Mediators in Precision-Cut Slices of the Murine Lung

Abstract

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