Highlights d Palmitoylated APP (palAPP) is enriched in mitochondrialassociated ER membranes (MAMs) d Upregulation of MAM increases cell surface palAPP levels and axonal Ab generation d Downregulation of MAM attenuates cell surface palAPP levels and axonal Ab generation

Summary This protocol describes the differentiation of human neural progenitor cells (hNPCs) in a microfluidic device containing a thin 3D matrix with two separate chambers, enabling a cleaner separation between axons and soma/bulk neurons. We have used this technique to study how mitochondria-associated ER membranes (MAMs) regulate the generation of somal and axonal amyloid β (Aβ) in FAD hNPCs, a cellular model of Alzheimer’s disease. This protocol also details the quantification of Aβ molecules and isolation of pure axons via axotomy. For complete details on the use and execution of this profile, please refer to Bhattacharyya et al. (2021) .