Enrichment of presynaptic and postsynaptic markers by size-based gating analysis of synaptosome preparations from rat and human cortex

Background: Synapse regions in the brain are difficult to isolate and study; resealed nerve terminals (synapto-somes) are a widely used in vitro system for the study of neurotransmission, but nonsynaptosomal elements in the homogenate complicate data interpretation. With the goal of quantitative analysis of pathways leading to synapse loss in neurodegenerative disease, we have developed a method that allows focus on the intact synaptosomes within a crude synaptosomal preparation by gating the largest particles based on forward angle light scatter (FSC). Methods: Crude synaptosomal fractions (P-2) were pre-pared and labeled with a viability dye (calcein AM), a presynaptic marker (SNAP-25), and a postsynaptic marker (PSD-95). Forward scatter gates based on size standard