Streamlined isolation of enriched axoplasm from primary dissociated neuronal cultures

Axons are highly specialized neuronal compartments that rely on local molecular regulation to support growth, maintenance, and synaptic function. The cytoplasmic contents of axons, collectively referred to as the axoplasm, constitute a biochemically distinct domain whose composition is central to neuronal development and disease. However, biochemical analysis of axoplasm has been technically challenging, particularly in dissociated neuronal cultures, where axons and somata are intermingled and axonal material is limited. As a result, most axoplasm-focused studies have relied on explant-based or peripheral neuron models, restricting direct biochemical access to axons from dissociated central nervous system neurons. Here, we describe a simple and robust culture-based approach that enables efficient physical separation of somata and axons from primary dissociated neuronal cultures and allows the recovery of enriched axoplasm fractions. Using a custom reaggregation device, neurons reorganize into compact somatic clusters that extend radially oriented axons, facilitating reliable manual separation of somatic and axonal material. Morphological and immunofluorescence analyses demonstrate robust neuronal polarization and effective spatial segregation of somatic and axonal components. Biochemical and molecular validation reveals that axonal fractions are enriched in axonal proteins and contain bona fide axon-associated transcripts, while being depleted of nuclear markers and genomic DNA, providing a practical and accessible method for axoplasm isolation from dissociated neuronal cultures. By enabling biochemical analysis of axonal material from neuron types that are otherwise difficult to access, this method expands the experimental toolkit for studying axon-specific molecular mechanisms in neuronal development, plasticity, and disease.