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Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain
Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine relea...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MyJove Corporation
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4828148/ https://www.ncbi.nlm.nih.gov/pubmed/26967380 http://dx.doi.org/10.3791/53658 |
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author | Pösel, Claudia Möller, Karoline Boltze, Johannes Wagner, Daniel-Christoph Weise, Gesa |
author_facet | Pösel, Claudia Möller, Karoline Boltze, Johannes Wagner, Daniel-Christoph Weise, Gesa |
author_sort | Pösel, Claudia |
collection | PubMed |
description | Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed. Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair. |
format | Online Article Text |
id | pubmed-4828148 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | MyJove Corporation |
record_format | MEDLINE/PubMed |
spelling | pubmed-48281482016-04-22 Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain Pösel, Claudia Möller, Karoline Boltze, Johannes Wagner, Daniel-Christoph Weise, Gesa J Vis Exp Immunology Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed. Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair. MyJove Corporation 2016-02-12 /pmc/articles/PMC4828148/ /pubmed/26967380 http://dx.doi.org/10.3791/53658 Text en Copyright © 2016, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/ |
spellingShingle | Immunology Pösel, Claudia Möller, Karoline Boltze, Johannes Wagner, Daniel-Christoph Weise, Gesa Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain |
title | Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain |
title_full | Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain |
title_fullStr | Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain |
title_full_unstemmed | Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain |
title_short | Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain |
title_sort | isolation and flow cytometric analysis of immune cells from the ischemic mouse brain |
topic | Immunology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4828148/ https://www.ncbi.nlm.nih.gov/pubmed/26967380 http://dx.doi.org/10.3791/53658 |
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