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Quantifying mitochondrial redox and bilirubin content in intact primary hepatocytes of obese mice using fluorescent reporters
Assessing the physiological role of H(2)O(2) requires sensitive techniques to quantify H(2)O(2) and antioxidants in live cells. Here, we present a protocol to assess the mitochondrial redox state and unconjugated bilirubin levels in intact live primary hepatocytes from obese mice. We described steps...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10336327/ https://www.ncbi.nlm.nih.gov/pubmed/37393613 http://dx.doi.org/10.1016/j.xpro.2023.102408 |
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author | Belmas, Thomas Liesa, Marc Shum, Michaël |
author_facet | Belmas, Thomas Liesa, Marc Shum, Michaël |
author_sort | Belmas, Thomas |
collection | PubMed |
description | Assessing the physiological role of H(2)O(2) requires sensitive techniques to quantify H(2)O(2) and antioxidants in live cells. Here, we present a protocol to assess the mitochondrial redox state and unconjugated bilirubin levels in intact live primary hepatocytes from obese mice. We described steps to quantify H(2)O(2), GSSG/GSH, and bilirubin content in the mitochondrial matrix and the cytosol using the fluorescent reporters roGFP2-ORP1, GRX1-roGFP2, and UnaG, respectively. We detail hepatocyte isolation, plating, and transduction and live-cell imaging using a high-content imaging reader. For complete details on the use and execution of this protocol, please refer to Shum et al.(1) |
format | Online Article Text |
id | pubmed-10336327 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-103363272023-07-13 Quantifying mitochondrial redox and bilirubin content in intact primary hepatocytes of obese mice using fluorescent reporters Belmas, Thomas Liesa, Marc Shum, Michaël STAR Protoc Protocol Assessing the physiological role of H(2)O(2) requires sensitive techniques to quantify H(2)O(2) and antioxidants in live cells. Here, we present a protocol to assess the mitochondrial redox state and unconjugated bilirubin levels in intact live primary hepatocytes from obese mice. We described steps to quantify H(2)O(2), GSSG/GSH, and bilirubin content in the mitochondrial matrix and the cytosol using the fluorescent reporters roGFP2-ORP1, GRX1-roGFP2, and UnaG, respectively. We detail hepatocyte isolation, plating, and transduction and live-cell imaging using a high-content imaging reader. For complete details on the use and execution of this protocol, please refer to Shum et al.(1) Elsevier 2023-07-01 /pmc/articles/PMC10336327/ /pubmed/37393613 http://dx.doi.org/10.1016/j.xpro.2023.102408 Text en © 2023 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Protocol Belmas, Thomas Liesa, Marc Shum, Michaël Quantifying mitochondrial redox and bilirubin content in intact primary hepatocytes of obese mice using fluorescent reporters |
title | Quantifying mitochondrial redox and bilirubin content in intact primary hepatocytes of obese mice using fluorescent reporters |
title_full | Quantifying mitochondrial redox and bilirubin content in intact primary hepatocytes of obese mice using fluorescent reporters |
title_fullStr | Quantifying mitochondrial redox and bilirubin content in intact primary hepatocytes of obese mice using fluorescent reporters |
title_full_unstemmed | Quantifying mitochondrial redox and bilirubin content in intact primary hepatocytes of obese mice using fluorescent reporters |
title_short | Quantifying mitochondrial redox and bilirubin content in intact primary hepatocytes of obese mice using fluorescent reporters |
title_sort | quantifying mitochondrial redox and bilirubin content in intact primary hepatocytes of obese mice using fluorescent reporters |
topic | Protocol |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10336327/ https://www.ncbi.nlm.nih.gov/pubmed/37393613 http://dx.doi.org/10.1016/j.xpro.2023.102408 |
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