Separation, isolation, and membrane lipid profiling of selectively vulnerable and resilient neurons

Certain neurons are more susceptible to pathogenic stressors and degenerate, while others remain resilient in neurodegenerative diseases. However, the mechanisms underlying this cell-to-cell heterogeneity in neuronal vulnerability remain unclear, primarily due to challenges in isolating vulnerable and resilient neurons to characterize their properties. In this study, we report the development of a novel cell-labeling method utilizing two cell-impermeable dyes to identify vulnerable and resilient neurons in culture, which are subsequently separated and isolated via fluorescence-activated cell sorting (FACS). As a proof-of-concept of a mechanistic study, we conducted lipidomic analysis to determine whether the lipid composition of membranes differs between vulnerable and resilient neurons to oxidative stress. This simple but unique method sets the groundwork for investigating the molecular underpinnings of selective neuronal vulnerability, which could inform new diagnostic strategies and therapeutic targets for neurodegenerative diseases.