Fast and efficient synaptosome isolation and post-synaptic density enrichment from hiPSC-motor neurons by biochemical sub-cellular fractionation

• Published in: STAR protocols Vol. 4; no. 1; p. 102061
• Main Authors: Rajkumar, Sandeep, Böckers, Tobias M., Catanese, Alberto
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.03.2023; Elsevier
• Subjects: Cell culture; Cell separation/fractionation; Neuroscience; Protocol; Stem Cells; Stem Cells; Cell separation/fractionation; Cell culture; Neuroscience
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102061

We describe here a time-efficient, in-house protocol for synaptosome isolation and enrichment of the post-synaptic density (PSD) from hiPSC-derived motor neurons. By using biochemical sub-cellular fractionation, the crude synaptosome is first isolated from the cytosol and is then further separated into the synaptic cytosol and the enriched PSD fraction. The protocol can also potentially be adapted to other hiPSC-derived neuronal types, with necessary changes made to cell seeding density and buffer volumes. [Display omitted] •Fast and efficient synaptosome isolation from hiPSC-derived motor neurons•Subsequent fractionation into synaptic cytosol and highly enriched PSD•Confirmation of percentage enrichment by standard Western blotting•Substantial protein quality and concentration for high-throughput proteomics Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. We describe here a time-efficient, in-house protocol for synaptosome isolation and enrichment of the post-synaptic density (PSD) from hiPSC-derived motor neurons. By using biochemical sub-cellular fractionation, the crude synaptosome is first isolated from the cytosol and is then further separated into the synaptic cytosol and the enriched PSD fraction. The protocol can also potentially be adapted to other hiPSC-derived neuronal types, with necessary changes made to cell seeding density and buffer volumes.