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

Miriam E. Korsgen; Congxin Sun; Elena Seranova; Malgorzata Zatyka; Dewi Astuti; Tetsushi Kataura; Timothy Barrett; Viktor I. Korolchuk; Sovan Sarkar

STAR Protocols (Sep 2023)

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

Miriam E. Korsgen,
Congxin Sun,
Elena Seranova,
Malgorzata Zatyka,
Dewi Astuti,
Tetsushi Kataura,
Timothy Barrett,
Viktor I. Korolchuk,
Sovan Sarkar

Affiliations

Miriam E. Korsgen: Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Congxin Sun: Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK; Corresponding author
Elena Seranova: Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Malgorzata Zatyka: Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Dewi Astuti: Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Tetsushi Kataura: Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE4 5PL, UK
Timothy Barrett: Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK; Department of Endocrinology, Birmingham Women’s and Children’s Hospital, Steelhouse Lane, Birmingham B4 6NH, UK
Viktor I. Korolchuk: Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE4 5PL, UK; Corresponding author
Sovan Sarkar: Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK; Corresponding author

Journal volume & issue: Vol. 4, no. 3
p. 102529

Abstract

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Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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