Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons

Autophagy, a catabolic process governing cellular and energy homeostasis, is essential for cell survival and human health. Here, we present a protocol for generating autophagy-deficient (ATG5(−/−)) human neurons from human embryonic stem cell (hESC)-derived neural precursors. We describe steps for analyzing loss of autophagy by immunoblotting. We then detail analysis of cell death by luminescence-based cytotoxicity assay and fluorescence-based TUNEL staining. This hESC-based experimental platform provides a genetic knockout model for undertaking autophagy studies relevant to human biology. For complete details on the use and execution of this protocol, please refer to Sun et al. (2023).(1)