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

Lookup NU author(s): Dr Tetsushi Kataura, Dr Viktor Korolchuk

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).

Abstract

© 2023 The Author(s)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

Publication metadata

Author(s): Korsgen ME, Sun C, Seranova E, Zatyka M, Astuti D, Kataura T, Barrett T, Korolchuk VI, Sarkar S

Publication type: Article

Publication status: Published

Journal: STAR Protocols

Year: 2023

Volume: 4

Issue: 3

Print publication date: 15/09/2023

Online publication date: 25/08/2023

Acceptance date: 02/04/2018

Date deposited: 13/11/2023

ISSN (electronic): 2666-1667

Publisher: Cell Press

URL: https://doi.org/10.1016/j.xpro.2023.102529

DOI: 10.1016/j.xpro.2023.102529

PubMed id: 37624702

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