Activated producing HOCL neutrophils revealed by flow cytometry and confocal microscopy with celestine blue B
Hypochlorous acid (HOCl) is produced with myeloperoxidase (MPO) in activated neutrophils. To assay MPO activity we chose a HOCl-selective dye i.e. celestine blue B (CB) that changes into pink glycol after oxidation. Our aim was to elaborate fluorescent methods of measuring HOCl production with activ...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | Russian |
| Published: |
Scientific Сentre for Family Health and Human Reproduction Problems
2016-05-01
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| Series: | Acta Biomedica Scientifica |
| Subjects: | |
| Online Access: | https://www.actabiomedica.ru/jour/article/view/194 |
| _version_ | 1797237417881632768 |
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| author | S. O. Kozlov I. V. Kudryavtsev N. A. Grudinina V. A. Kostevich O. M. Panasenko A. V. Sokolov V. B. Vasilyev |
| author_facet | S. O. Kozlov I. V. Kudryavtsev N. A. Grudinina V. A. Kostevich O. M. Panasenko A. V. Sokolov V. B. Vasilyev |
| author_sort | S. O. Kozlov |
| collection | DOAJ |
| description | Hypochlorous acid (HOCl) is produced with myeloperoxidase (MPO) in activated neutrophils. To assay MPO activity we chose a HOCl-selective dye i.e. celestine blue B (CB) that changes into pink glycol after oxidation. Our aim was to elaborate fluorescent methods of measuring HOCl production with activated neutrophils. Scanning CB fluorescence before and after its reaction with HOCl revealed activation and maximum emission, at 487 and 578 nm respectively, which are specific for oxidized product. Activated with Phorbol 12-myristate 13-acetate (PMA), the neutrophils were incubated with MPO inhibitor, 4-aminobenzoic acid hydrazide, which decreased fluorescence intensity (activation 487 nm, emission 578 nm) as compared with inhibitor-free samples. By confocal microscopy method we obtained images of CB- and DAPI-stained neutrophils. Neutrophil extracellular traps (NET) are clearly visible: DAP1 staining of multiple DNA bands co-localizes with fluorescence of oxidized CB. Flow cytometry showed that intensity of neutrophils activated by 50 nM PMA was 5 times higher (p < 0.05) than in PMA-free cells. |
| first_indexed | 2024-03-08T12:19:03Z |
| format | Article |
| id | doaj.art-8b61966afefd4a95aef2fec02fb35855 |
| institution | Directory Open Access Journal |
| issn | 2541-9420 2587-9596 |
| language | Russian |
| last_indexed | 2024-04-24T17:19:25Z |
| publishDate | 2016-05-01 |
| publisher | Scientific Сentre for Family Health and Human Reproduction Problems |
| record_format | Article |
| series | Acta Biomedica Scientifica |
| spelling | doaj.art-8b61966afefd4a95aef2fec02fb358552024-03-28T07:49:15ZrusScientific Сentre for Family Health and Human Reproduction ProblemsActa Biomedica Scientifica2541-94202587-95962016-05-0113(2)869110.12737/article_590823a4895537.04307905194Activated producing HOCL neutrophils revealed by flow cytometry and confocal microscopy with celestine blue BS. O. Kozlov0I. V. Kudryavtsev1N. A. Grudinina2V. A. Kostevich3O. M. Panasenko4A. V. Sokolov5V. B. Vasilyev6Institute of Experimental MedicineInstitute of Experimental Medicine; Pavlov First Saint Petersburg State Medical UniversityInstitute of Experimental MedicineInstitute of Experimental Medicine; Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological AgencyFederal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological AgencyInstitute of Experimental Medicine; Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological AgencyInstitute of Experimental Medicine; Saint-Petersburg State UniversityHypochlorous acid (HOCl) is produced with myeloperoxidase (MPO) in activated neutrophils. To assay MPO activity we chose a HOCl-selective dye i.e. celestine blue B (CB) that changes into pink glycol after oxidation. Our aim was to elaborate fluorescent methods of measuring HOCl production with activated neutrophils. Scanning CB fluorescence before and after its reaction with HOCl revealed activation and maximum emission, at 487 and 578 nm respectively, which are specific for oxidized product. Activated with Phorbol 12-myristate 13-acetate (PMA), the neutrophils were incubated with MPO inhibitor, 4-aminobenzoic acid hydrazide, which decreased fluorescence intensity (activation 487 nm, emission 578 nm) as compared with inhibitor-free samples. By confocal microscopy method we obtained images of CB- and DAPI-stained neutrophils. Neutrophil extracellular traps (NET) are clearly visible: DAP1 staining of multiple DNA bands co-localizes with fluorescence of oxidized CB. Flow cytometry showed that intensity of neutrophils activated by 50 nM PMA was 5 times higher (p < 0.05) than in PMA-free cells.https://www.actabiomedica.ru/jour/article/view/194hoclhoclhypochlorous acidmyeloperoxidasecelestine blue bneutrophilconfocal microscopyflow cytometryfluorescence |
| spellingShingle | S. O. Kozlov I. V. Kudryavtsev N. A. Grudinina V. A. Kostevich O. M. Panasenko A. V. Sokolov V. B. Vasilyev Activated producing HOCL neutrophils revealed by flow cytometry and confocal microscopy with celestine blue B Acta Biomedica Scientifica hocl hocl hypochlorous acid myeloperoxidase celestine blue b neutrophil confocal microscopy flow cytometry fluorescence |
| title | Activated producing HOCL neutrophils revealed by flow cytometry and confocal microscopy with celestine blue B |
| title_full | Activated producing HOCL neutrophils revealed by flow cytometry and confocal microscopy with celestine blue B |
| title_fullStr | Activated producing HOCL neutrophils revealed by flow cytometry and confocal microscopy with celestine blue B |
| title_full_unstemmed | Activated producing HOCL neutrophils revealed by flow cytometry and confocal microscopy with celestine blue B |
| title_short | Activated producing HOCL neutrophils revealed by flow cytometry and confocal microscopy with celestine blue B |
| title_sort | activated producing hocl neutrophils revealed by flow cytometry and confocal microscopy with celestine blue b |
| topic | hocl hocl hypochlorous acid myeloperoxidase celestine blue b neutrophil confocal microscopy flow cytometry fluorescence |
| url | https://www.actabiomedica.ru/jour/article/view/194 |
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