Capturing Microglia-Derived Extracellular Vesicles in Acute Ischemic Stroke: A Bead-Based Flow Cytometry Approach

Abstract

Objective: Recent findings suggest that microglia, the resident immune cells of the central nervous system (CNS), respond to stimuli in the internal environment by dynamically changing their phenotype and releasing extracellular vesicles (EVs) that contribute to neurorepair and neuroprotection after stroke. We investigated the isolation of EVs secreted by microglia (MDEVs) from plasma samples of acute ischemic stroke (IS) patients.

Methods: EVs were isolated from patients’ plasma at three key time points—24 hours, 7 days, and one month following symptom onset—using the ExoQuick® ULTRA EV precipitation kit. Subpopulations of MDEVs were purified based on common EV protein markers like CD81, CD63, and CD9 tetraspanins, as well as transmembrane protein 119 (TMEM119), which specifically indicates microglia, through the Basic Exo-Flow Capture kit. The obtained antibody-coupled bead-associated particles were then labeled with Exo-FITC and analyzed with the BD FACSAria™ III flow cytometer.

Results: Flow cytometry analysis confirmed a pure and highly enriched MDEV suspension appropriate for a variety of downstream applications in future research. Fluorescein isothiocyanate (FITC) median fluorescence intensities (MFI) remained consistent across all evaluated post-stroke time points, indicating that similar amounts of EVs were recovered from each patient with uniform capture efficiency. However, compared to controls, FITC-MFIs were significantly higher in IS patients.

Conclusions: Studying EV populations is challenging due to their heterogeneity. This MDEV purification protocol could provide a new, noninvasive method for CNS monitoring, making it a valuable tool for biomarker discovery in the diagnosis, prognosis, and treatment of IS.