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Optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry
The limited availability of rapid and reliable flow cytometry-based assays for ex vivo quantification of autophagy has hampered their clinical applications for studies of diseases pathogenesis or for the implementation of autophagy-targeting therapies. To this aim, we modified and improved the proto...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PAGEPress Publications, Pavia, Italy
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610717/ https://www.ncbi.nlm.nih.gov/pubmed/31243942 http://dx.doi.org/10.4081/ejh.2019.3044 |
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author | Alonzi, Tonino Petruccioli, Elisa Vanini, Valentina Fimia, Gian Maria Goletti, Delia |
author_facet | Alonzi, Tonino Petruccioli, Elisa Vanini, Valentina Fimia, Gian Maria Goletti, Delia |
author_sort | Alonzi, Tonino |
collection | PubMed |
description | The limited availability of rapid and reliable flow cytometry-based assays for ex vivo quantification of autophagy has hampered their clinical applications for studies of diseases pathogenesis or for the implementation of autophagy-targeting therapies. To this aim, we modified and improved the protocol of a commercial kit developed for quantifying the microtubule-associated protein 1A/1B light chain 3B (LC3), the most reliable marker for autophagosomes currently available. The protocol modifications were set up measuring the autophagic flux in neoplastic (THP-1 cells) and primary cells (peripheral blood mononuclear cells; PBMC) of healthy donors. Moreover, PBMC of active tuberculosis (TB) patients were stimulated with the Mycobacterium tuberculosis purified protein derivatives or infected with live Mycobacterium bovis bacillus Calmette-Guerin (BCG). We found that the baseline median fluorescent intensity (MFI) of THP-1 cells changed depending on the time of sample acquisition to the flow cytometer. To solve this problem, a fixation step was introduced in different stages of the assay’s protocol, obtaining more reproducible and sensitive results when a post-LC3-staining fixation was performed, in either THP-1 cells or PBMC. Furthermore, since we found that results are influenced by the type and the dose of the lysosome inhibitor used, the best dose of Chloroquine for LC3 accumulation were set up in either THP-1 cells or PBMC. Finally, applying these experimental settings, we measured the autophagic flux in CD14(+) cells from active TB patients’ PBMC upon BCG infection. In conclusion, our data indicate that the protocol modifications here described improve the stability and accuracy of a flow cytometry-based assay for the evaluation of autophagy, thus assuring more standardised cell analyses. |
format | Online Article Text |
id | pubmed-6610717 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | PAGEPress Publications, Pavia, Italy |
record_format | MEDLINE/PubMed |
spelling | pubmed-66107172019-07-24 Optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry Alonzi, Tonino Petruccioli, Elisa Vanini, Valentina Fimia, Gian Maria Goletti, Delia Eur J Histochem Original Paper The limited availability of rapid and reliable flow cytometry-based assays for ex vivo quantification of autophagy has hampered their clinical applications for studies of diseases pathogenesis or for the implementation of autophagy-targeting therapies. To this aim, we modified and improved the protocol of a commercial kit developed for quantifying the microtubule-associated protein 1A/1B light chain 3B (LC3), the most reliable marker for autophagosomes currently available. The protocol modifications were set up measuring the autophagic flux in neoplastic (THP-1 cells) and primary cells (peripheral blood mononuclear cells; PBMC) of healthy donors. Moreover, PBMC of active tuberculosis (TB) patients were stimulated with the Mycobacterium tuberculosis purified protein derivatives or infected with live Mycobacterium bovis bacillus Calmette-Guerin (BCG). We found that the baseline median fluorescent intensity (MFI) of THP-1 cells changed depending on the time of sample acquisition to the flow cytometer. To solve this problem, a fixation step was introduced in different stages of the assay’s protocol, obtaining more reproducible and sensitive results when a post-LC3-staining fixation was performed, in either THP-1 cells or PBMC. Furthermore, since we found that results are influenced by the type and the dose of the lysosome inhibitor used, the best dose of Chloroquine for LC3 accumulation were set up in either THP-1 cells or PBMC. Finally, applying these experimental settings, we measured the autophagic flux in CD14(+) cells from active TB patients’ PBMC upon BCG infection. In conclusion, our data indicate that the protocol modifications here described improve the stability and accuracy of a flow cytometry-based assay for the evaluation of autophagy, thus assuring more standardised cell analyses. PAGEPress Publications, Pavia, Italy 2019-06-26 /pmc/articles/PMC6610717/ /pubmed/31243942 http://dx.doi.org/10.4081/ejh.2019.3044 Text en ©Copyright T. Alonzi et al., 2019 http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (by-nc 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. |
spellingShingle | Original Paper Alonzi, Tonino Petruccioli, Elisa Vanini, Valentina Fimia, Gian Maria Goletti, Delia Optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry |
title | Optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry |
title_full | Optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry |
title_fullStr | Optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry |
title_full_unstemmed | Optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry |
title_short | Optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry |
title_sort | optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry |
topic | Original Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610717/ https://www.ncbi.nlm.nih.gov/pubmed/31243942 http://dx.doi.org/10.4081/ejh.2019.3044 |
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