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...

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Autores principales:	S. O. Kozlov, I. V. Kudryavtsev, N. A. Grudinina, V. A. Kostevich, O. M. Panasenko, A. V. Sokolov, V. B. Vasilyev
Formato:	article
Lenguaje:	RU
Publicado:	Scientific Сentre for Family Health and Human Reproduction Problems 2016
Materias:	hocl hypochlorous acid myeloperoxidase celestine blue b neutrophil confocal microscopy flow cytometry fluorescence Science Q
Acceso en línea:	https://doaj.org/article/8b61966afefd4a95aef2fec02fb35855
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spelling	oai:doaj.org-article:8b61966afefd4a95aef2fec02fb358552021-11-23T06:14:34ZActivated producing HOCL neutrophils revealed by flow cytometry and confocal microscopy with celestine blue B2541-94202587-959610.12737/article_590823a4895537.04307905https://doaj.org/article/8b61966afefd4a95aef2fec02fb358552016-05-01T00:00:00Zhttps://www.actabiomedica.ru/jour/article/view/194https://doaj.org/toc/2541-9420https://doaj.org/toc/2587-9596Hypochlorous 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.S. O. KozlovI. V. KudryavtsevN. A. GrudininaV. A. KostevichO. M. PanasenkoA. V. SokolovV. B. VasilyevScientific Сentre for Family Health and Human Reproduction Problemsarticlehoclhoclhypochlorous acidmyeloperoxidasecelestine blue bneutrophilconfocal microscopyflow cytometryfluorescenceScienceQRUActa Biomedica Scientifica, Vol 1, Iss 3(2), Pp 86-91 (2016)
institution	DOAJ
collection	DOAJ
language	RU
topic	hocl hocl hypochlorous acid myeloperoxidase celestine blue b neutrophil confocal microscopy flow cytometry fluorescence Science Q
spellingShingle	hocl hocl hypochlorous acid myeloperoxidase celestine blue b neutrophil confocal microscopy flow cytometry fluorescence Science Q 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
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.
format	article
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
title	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_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_sort	activated producing hocl neutrophils revealed by flow cytometry and confocal microscopy with celestine blue b
publisher	Scientific Сentre for Family Health and Human Reproduction Problems
publishDate	2016
url	https://doaj.org/article/8b61966afefd4a95aef2fec02fb35855
work_keys_str_mv	AT sokozlov activatedproducinghoclneutrophilsrevealedbyflowcytometryandconfocalmicroscopywithcelestineblueb AT ivkudryavtsev activatedproducinghoclneutrophilsrevealedbyflowcytometryandconfocalmicroscopywithcelestineblueb AT nagrudinina activatedproducinghoclneutrophilsrevealedbyflowcytometryandconfocalmicroscopywithcelestineblueb AT vakostevich activatedproducinghoclneutrophilsrevealedbyflowcytometryandconfocalmicroscopywithcelestineblueb AT ompanasenko activatedproducinghoclneutrophilsrevealedbyflowcytometryandconfocalmicroscopywithcelestineblueb AT avsokolov activatedproducinghoclneutrophilsrevealedbyflowcytometryandconfocalmicroscopywithcelestineblueb AT vbvasilyev activatedproducinghoclneutrophilsrevealedbyflowcytometryandconfocalmicroscopywithcelestineblueb
_version_	1718417080445829120

Activated producing HOCL neutrophils revealed by flow cytometry and confocal microscopy with celestine blue B

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