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 a...

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Autores principales: Korsgen, Miriam E., Sun, Congxin, Seranova, Elena, Zatyka, Malgorzata, Astuti, Dewi, Kataura, Tetsushi, Barrett, Timothy, Korolchuk, Viktor I., Sarkar, Sovan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10474488/
https://www.ncbi.nlm.nih.gov/pubmed/37624702
http://dx.doi.org/10.1016/j.xpro.2023.102529
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author Korsgen, Miriam E.
Sun, Congxin
Seranova, Elena
Zatyka, Malgorzata
Astuti, Dewi
Kataura, Tetsushi
Barrett, Timothy
Korolchuk, Viktor I.
Sarkar, Sovan
author_facet Korsgen, Miriam E.
Sun, Congxin
Seranova, Elena
Zatyka, Malgorzata
Astuti, Dewi
Kataura, Tetsushi
Barrett, Timothy
Korolchuk, Viktor I.
Sarkar, Sovan
author_sort Korsgen, Miriam E.
collection PubMed
description 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)
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spelling pubmed-104744882023-09-03 Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons Korsgen, Miriam E. Sun, Congxin Seranova, Elena Zatyka, Malgorzata Astuti, Dewi Kataura, Tetsushi Barrett, Timothy Korolchuk, Viktor I. Sarkar, Sovan STAR Protoc Protocol 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) Elsevier 2023-08-25 /pmc/articles/PMC10474488/ /pubmed/37624702 http://dx.doi.org/10.1016/j.xpro.2023.102529 Text en © 2023 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Korsgen, Miriam E.
Sun, Congxin
Seranova, Elena
Zatyka, Malgorzata
Astuti, Dewi
Kataura, Tetsushi
Barrett, Timothy
Korolchuk, Viktor I.
Sarkar, Sovan
Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons
title Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons
title_full Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons
title_fullStr Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons
title_full_unstemmed Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons
title_short Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons
title_sort analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10474488/
https://www.ncbi.nlm.nih.gov/pubmed/37624702
http://dx.doi.org/10.1016/j.xpro.2023.102529
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