New Laboratory Protocol to Determine the Oxidative Stress Profile of Human Nasal Epithelial Cells Using Flow Cytometry
Several studies have shown the importance of oxidative stress (OS) in respiratory disease pathogenesis. It has been reported that the nasal epithelium may act as a surrogate for the bronchial epithelium in several respiratory diseases involving OS. However, the sample yields obtained from nasal biop...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-03-01
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| Series: | Journal of Clinical Medicine |
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| Online Access: | https://www.mdpi.com/2077-0383/10/6/1172 |
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| author | Ana Reula Daniel Pellicer Silvia Castillo María Magallón Miguel Armengot Guadalupe Herrera José-Enrique O’Connor Lucía Bañuls María Mercedes Navarro-García Amparo Escribano Francisco Dasí |
| author_facet | Ana Reula Daniel Pellicer Silvia Castillo María Magallón Miguel Armengot Guadalupe Herrera José-Enrique O’Connor Lucía Bañuls María Mercedes Navarro-García Amparo Escribano Francisco Dasí |
| author_sort | Ana Reula |
| collection | DOAJ |
| description | Several studies have shown the importance of oxidative stress (OS) in respiratory disease pathogenesis. It has been reported that the nasal epithelium may act as a surrogate for the bronchial epithelium in several respiratory diseases involving OS. However, the sample yields obtained from nasal biopsies are modest, limiting the number of parameters that can be determined. Flow cytometry has been widely used to evaluate cellular OS profiles. It has the advantage that analyses can be performed using a small amount of sample. Therefore, we aimed to set up a new method based on flow cytometry to assess the oxidative profile of human nasal epithelial cells which could be used in research on respiratory diseases. Levels of total nitric oxide, superoxide anion, peroxynitrite, and intracellular peroxides were measured. Reduced thiol levels, such as antioxidant-reduced glutathione and oxidative damaged lipids and proteins, were also analysed. The intracellular calcium levels, plasma membrane potential, apoptosis, and percentage of live cells were also studied. Finally, a strategy to evaluate the mitochondrial function, including mitochondrial hydrogen peroxide, superoxide anion, mitochondrial mass, and membrane potential, was set up. Using small amounts of sample and a non-invasive sampling technique, the described method enables the measurement of a comprehensive set of OS parameters in nasal epithelial cells, which could be useful in research on respiratory diseases. |
| first_indexed | 2024-03-10T13:20:20Z |
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| id | doaj.art-6f68b7a6f4144e968b61a1942b56a6ea |
| institution | Directory Open Access Journal |
| issn | 2077-0383 |
| language | English |
| last_indexed | 2024-03-10T13:20:20Z |
| publishDate | 2021-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Clinical Medicine |
| spelling | doaj.art-6f68b7a6f4144e968b61a1942b56a6ea2023-11-21T10:03:52ZengMDPI AGJournal of Clinical Medicine2077-03832021-03-01106117210.3390/jcm10061172New Laboratory Protocol to Determine the Oxidative Stress Profile of Human Nasal Epithelial Cells Using Flow CytometryAna Reula0Daniel Pellicer1Silvia Castillo2María Magallón3Miguel Armengot4Guadalupe Herrera5José-Enrique O’Connor6Lucía Bañuls7María Mercedes Navarro-García8Amparo Escribano9Francisco Dasí10Department of Physiology, School of Medicine, University of Valencia, Avda. Blasco Ibáñez, 17, 46010 Valencia, SpainDepartment of Physiology, School of Medicine, University of Valencia, Avda. Blasco Ibáñez, 17, 46010 Valencia, SpainRare Respiratory Diseases Research Group, IIS INCLIVA, Fundación Investigación Hospital Clínico Valencia, Avda. Menéndez y Pelayo, 4, 46010 Valencia, SpainDepartment of Physiology, School of Medicine, University of Valencia, Avda. Blasco Ibáñez, 17, 46010 Valencia, SpainDepartment of Surgery, School of Medicine, University of Valencia, Avda. Blasco Ibáñez, 17, 46010 Valencia, SpainFlow Cytometry Unit, IIS INCLIVA, Fundación Investigación Hospital Clínico Valencia, Avda. Menéndez y Pelayo, 4, 46010 Valencia, SpainDepartment of Biochemistry, School of Medicine, University of Valencia, Avda. Blasco Ibáñez, 17, 46010 Valencia, SpainDepartment of Physiology, School of Medicine, University of Valencia, Avda. Blasco Ibáñez, 17, 46010 Valencia, SpainRare Respiratory Diseases Research Group, IIS INCLIVA, Fundación Investigación Hospital Clínico Valencia, Avda. Menéndez y Pelayo, 4, 46010 Valencia, SpainRare Respiratory Diseases Research Group, IIS INCLIVA, Fundación Investigación Hospital Clínico Valencia, Avda. Menéndez y Pelayo, 4, 46010 Valencia, SpainDepartment of Physiology, School of Medicine, University of Valencia, Avda. Blasco Ibáñez, 17, 46010 Valencia, SpainSeveral studies have shown the importance of oxidative stress (OS) in respiratory disease pathogenesis. It has been reported that the nasal epithelium may act as a surrogate for the bronchial epithelium in several respiratory diseases involving OS. However, the sample yields obtained from nasal biopsies are modest, limiting the number of parameters that can be determined. Flow cytometry has been widely used to evaluate cellular OS profiles. It has the advantage that analyses can be performed using a small amount of sample. Therefore, we aimed to set up a new method based on flow cytometry to assess the oxidative profile of human nasal epithelial cells which could be used in research on respiratory diseases. Levels of total nitric oxide, superoxide anion, peroxynitrite, and intracellular peroxides were measured. Reduced thiol levels, such as antioxidant-reduced glutathione and oxidative damaged lipids and proteins, were also analysed. The intracellular calcium levels, plasma membrane potential, apoptosis, and percentage of live cells were also studied. Finally, a strategy to evaluate the mitochondrial function, including mitochondrial hydrogen peroxide, superoxide anion, mitochondrial mass, and membrane potential, was set up. Using small amounts of sample and a non-invasive sampling technique, the described method enables the measurement of a comprehensive set of OS parameters in nasal epithelial cells, which could be useful in research on respiratory diseases.https://www.mdpi.com/2077-0383/10/6/1172flow cytometryrare respiratory diseasesnasal epitheliumoxidative stressreactive oxygen species |
| spellingShingle | Ana Reula Daniel Pellicer Silvia Castillo María Magallón Miguel Armengot Guadalupe Herrera José-Enrique O’Connor Lucía Bañuls María Mercedes Navarro-García Amparo Escribano Francisco Dasí New Laboratory Protocol to Determine the Oxidative Stress Profile of Human Nasal Epithelial Cells Using Flow Cytometry Journal of Clinical Medicine flow cytometry rare respiratory diseases nasal epithelium oxidative stress reactive oxygen species |
| title | New Laboratory Protocol to Determine the Oxidative Stress Profile of Human Nasal Epithelial Cells Using Flow Cytometry |
| title_full | New Laboratory Protocol to Determine the Oxidative Stress Profile of Human Nasal Epithelial Cells Using Flow Cytometry |
| title_fullStr | New Laboratory Protocol to Determine the Oxidative Stress Profile of Human Nasal Epithelial Cells Using Flow Cytometry |
| title_full_unstemmed | New Laboratory Protocol to Determine the Oxidative Stress Profile of Human Nasal Epithelial Cells Using Flow Cytometry |
| title_short | New Laboratory Protocol to Determine the Oxidative Stress Profile of Human Nasal Epithelial Cells Using Flow Cytometry |
| title_sort | new laboratory protocol to determine the oxidative stress profile of human nasal epithelial cells using flow cytometry |
| topic | flow cytometry rare respiratory diseases nasal epithelium oxidative stress reactive oxygen species |
| url | https://www.mdpi.com/2077-0383/10/6/1172 |
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