Preparation and Characterization of Neutrophil Membrane-Fused Mitochondria (nMITO).

Mitochondrial transplantation is an emerging strategy for cellular repair, yet its efficiency is often limited by poor targeting and environmental instability. This protocol details the fabrication and comprehensive characterization of neutrophil membrane-fused mitochondria (nMITO), a hybrid organelle platform designed to combine the metabolic vigor of natural mitochondria with the targeting and anti-inflammatory properties of neutrophil membranes. We describe an optimized workflow for mouse heart mitochondrial isolation, lipopolysaccharide (LPS)-activated neutrophil membrane (NEM) extraction, and the subsequent sonication-mediated fusion process. Characterization techniques include dynamic light scattering (DLS) for size and zeta potential, transmission electron microscopy (TEM) for ultrastructural integrity, and bioenergetic assays [ATP synthesis and tetramethylrhodamine methyl ester (TMRM)-based membrane potential] to ensure functional preservation. Key features • The protocol provides a methodology for the isolation of neutrophil membranes from mouse bone marrow and mitochondria from the heart. • The protocol provides a methodology for the fabrication of neutrophil membrane-fused mitochondria (nMITO).