Large dense-core vesicles (LDcVs) contain a variety of neurotransmitters, proteins, and hormones such as biogenic amines and peptides, together with microRnAs (miRnAs). isolation of LDcVs is essential for functional studies including vesicle fusion, vesicle acidification, monoamine transport, and the miRNAs stored in LDCVs. Although several methods were reported for purifying LDCVs, the final fractions are significantly contaminated by other organelles, compromising biochemical characterization. Here we isolated LDCVs (chromaffin granules) with high yield and purity from bovine adrenal medulla. The fractionation protocol combines differential and continuous sucrose gradient centrifugation, allowing for reducing major contaminants such as mitochondria. Purified LDCVs show robust acidification by the endogenous V-ATPase and undergo SNARE-mediated fusion with artificial membranes. Interestingly, LDCVs contain specific miRNAs such as miR-375 and miR-375 is stabilized by protein complex against Rnase A. this protocol can be useful in research on the biological functions of LDcVs. Neurons communicate by neurotransmission, which involves vesicle fusion and neurotransmitter release 1,2. Synaptic vesicles and large dense-core vesicles (LDCVs) participate in different functions in the nervous system 3,4. Synaptic vesicles in the presynapse store classical neurotransmitters (e.g., glutamate, acetylcholine, GABA, and glycine), and mainly activate ion channels in the postsynaptic terminals, thereby transmitting electrical signals to the postsynapse. In contrast, LDCVs contain amines, neuropeptides, and other hormones that mainly stimulate G-protein-coupled receptors (GPCRs) to modulate synaptic responses 3-5. LDCVs are minor components, amounting to only 1~2% when compared to synaptic vesicles in the central nervous system 6-8 , but they are highly abundant in specialized brain regions (such as the hypothalamus), in dopaminergic neurons and in sympathetic neurons of the peripheral nervous system 6. Chromaffin cells are modified sympathetic ganglia of the sympathetic nervous system 7,8. Chromaffin cells are enriched with LDCVs, also called chromaffin granules, and therefore constitute a widely-used model system for in-depth study of LDCVs 9. LDCVs of chromaffin cells contain catecholamines (i.e., adrenaline, noradrenaline, and dopamine), peptides (e.g., encephalin and neuropeptide Y), and proteins (chromogranins, secretogranins) 10. Recently, we reported that LDCVs store microRNAs (miRNAs), which are released by vesicle fusion in response to stimulation, and miR-375 is the most abundant miRNA in LDCVs 11,12. A protocol to isolate LDCV from bovine adrenal medulla was reported in the 1950s 13 , then modified 14 to include step density gradient centrifugation in the late 1960s. This protocol was widely used in many studies and formed the basis for establishing the proteomics of LDCVs 15 , and for identifying their content including amines, peptides, and proteins released by LDCV exocytosis 10. However, the purity of LDCVs isolate...