Isolation and Flow Cytometric Assessment of Neuroimmune Interactions in a Mini-Stroke Murine Model.

Abstract

Stroke is a leading cause of death and long-term disability worldwide, with ischemic stroke representing the majority of cases. Following an ischemic stroke, resident and infiltrating immune cells become activated, contributing to further neuronal damage. However, the roles of the immune system in the pathology of ischemic stroke are not fully understood, largely due to the complex and dynamic regulation of immune responses in reaction to changes in the microenvironment during neuroinflammation. Therefore, it is essential to monitor and analyze the activation of resident and infiltrating immune cells over time after an ischemic stroke. In this study, we present a protocol for assessing the function of these immune cells following ischemic stroke using flow cytometric analysis in a mini-stroke model. We microdissect the infarcted brain tissue at specific time points and then dissociate it into a single-cell suspension using both mechanical and enzymatic methods. The cells are passed through a 70 µm cell strainer and labeled with fluorescently tagged antibody cocktails before being quantified by flow cytometric analysis. While this assay was specifically developed to investigate neuroimmune interactions after ischemic stroke, it can also be easily adapted to study neuroimmune mechanisms in other neurodegenerative diseases, such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease.