Isolation of Extracellular Vesicles from Sympathetic Neurons

Sympathetic neurons from TrkA^FLAG/FLAG animals were dissected and plated at 14,000 cells per microfluidic device as previously described^{36 (link),37 (link)}. At 6 DIV, 150 μL of complete media was added to the CB chamber and 100 μL of anti-FLAG-AF488 antibody (1:200) in complete media was added to the DA chamber. Conditioned media was collected from the CB chamber 15 h after the addition of the anti-FLAG-AF488 antibody and EVs were isolated. For inhibitor treatments, 100 μL of complete media containing inhibitors (LY294002 50 μM, U-73122 1 μM, or 0.1% v/v DMSO) was added to the CB chamber and 150 μL of anti-FLAG-AF488 antibody (1:200) in complete media was added the DA chamber. Conditioned media was collected from the CB chamber 15 h after the addition of the anti-FLAG-AF488 and inhibitors and EVs were isolated.