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Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix

Degranulation refers to the secretion of inflammatory mediators, such as histamine, serotonin, and proteases, that are stored within the granules of mast cells and that trigger allergic reactions. The amount of these released mediators has been measured biochemically using cell mass. To investigate...

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Detalles Bibliográficos
Autores principales: Yokawa, Satoru, Suzuki, Takahiro, Hayashi, Ayumi, Inouye, Satoshi, Inoh, Yoshikazu, Furuno, Tadahide
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048188/
https://www.ncbi.nlm.nih.gov/pubmed/30042943
http://dx.doi.org/10.3389/fcell.2018.00074
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author Yokawa, Satoru
Suzuki, Takahiro
Hayashi, Ayumi
Inouye, Satoshi
Inoh, Yoshikazu
Furuno, Tadahide
author_facet Yokawa, Satoru
Suzuki, Takahiro
Hayashi, Ayumi
Inouye, Satoshi
Inoh, Yoshikazu
Furuno, Tadahide
author_sort Yokawa, Satoru
collection PubMed
description Degranulation refers to the secretion of inflammatory mediators, such as histamine, serotonin, and proteases, that are stored within the granules of mast cells and that trigger allergic reactions. The amount of these released mediators has been measured biochemically using cell mass. To investigate degranulation in living single cells, fluorescence microscopy has traditionally been used to observe the disappearance of granules and the appearance of these discharged granules within the plasma membrane by membrane fusion and the movement of granules inside the cells. Here, we developed a method of video-rate bioluminescence imaging to directly detect degranulation from a single mast cell by measuring luminescence activity derived from the enzymatic reaction between Gaussia luciferase (GLase) and its substrate coelenterazine. The neuropeptide Y (NPY), which was reported to colocalize with serotonin in the secretory granules, fused to GLase (NPY-GLase) was efficiently expressed in rat basophilic leukemia (RBL-2H3) cells, a mast-cell line, using a preferred human codon-optimized gene. Bioluminescence imaging analysis of RBL-2H3 cells expressing NPY-GLase and adhered on a glass-bottomed dish showed that the luminescence signals from the resting cells were negligible, while the luminescence signals of the secreted NPY-GLase were repeatedly detected after the addition of an antigen. In addition, this imaging method was applicable for observing degranulation in RBL-2H3 cells that adhered to the extracellular matrix (ECM). These results indicated that video-rate bioluminescence imaging using GLase will be a useful tool for detecting degranulation in single mast cells adhered to a variety of ECM proteins.
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spelling pubmed-60481882018-07-24 Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix Yokawa, Satoru Suzuki, Takahiro Hayashi, Ayumi Inouye, Satoshi Inoh, Yoshikazu Furuno, Tadahide Front Cell Dev Biol Cell and Developmental Biology Degranulation refers to the secretion of inflammatory mediators, such as histamine, serotonin, and proteases, that are stored within the granules of mast cells and that trigger allergic reactions. The amount of these released mediators has been measured biochemically using cell mass. To investigate degranulation in living single cells, fluorescence microscopy has traditionally been used to observe the disappearance of granules and the appearance of these discharged granules within the plasma membrane by membrane fusion and the movement of granules inside the cells. Here, we developed a method of video-rate bioluminescence imaging to directly detect degranulation from a single mast cell by measuring luminescence activity derived from the enzymatic reaction between Gaussia luciferase (GLase) and its substrate coelenterazine. The neuropeptide Y (NPY), which was reported to colocalize with serotonin in the secretory granules, fused to GLase (NPY-GLase) was efficiently expressed in rat basophilic leukemia (RBL-2H3) cells, a mast-cell line, using a preferred human codon-optimized gene. Bioluminescence imaging analysis of RBL-2H3 cells expressing NPY-GLase and adhered on a glass-bottomed dish showed that the luminescence signals from the resting cells were negligible, while the luminescence signals of the secreted NPY-GLase were repeatedly detected after the addition of an antigen. In addition, this imaging method was applicable for observing degranulation in RBL-2H3 cells that adhered to the extracellular matrix (ECM). These results indicated that video-rate bioluminescence imaging using GLase will be a useful tool for detecting degranulation in single mast cells adhered to a variety of ECM proteins. Frontiers Media S.A. 2018-07-10 /pmc/articles/PMC6048188/ /pubmed/30042943 http://dx.doi.org/10.3389/fcell.2018.00074 Text en Copyright © 2018 Yokawa, Suzuki, Hayashi, Inouye, Inoh and Furuno. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cell and Developmental Biology
Yokawa, Satoru
Suzuki, Takahiro
Hayashi, Ayumi
Inouye, Satoshi
Inoh, Yoshikazu
Furuno, Tadahide
Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix
title Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix
title_full Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix
title_fullStr Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix
title_full_unstemmed Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix
title_short Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix
title_sort video-rate bioluminescence imaging of degranulation of mast cells attached to the extracellular matrix
topic Cell and Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048188/
https://www.ncbi.nlm.nih.gov/pubmed/30042943
http://dx.doi.org/10.3389/fcell.2018.00074
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