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Identification of primary retinal cells and ex vivo detection of proinflammatory molecules using flow cytometry

PURPOSE: Advances in the understanding of the pathogenesis of retinal disorders can be facilitated by a methodology to measure expression of proinflammatory molecules in various subsets of retinal cells. METHODS: We examined whether a multiparameter flow cytometric assay can be used to identify vari...

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Autores principales: Portillo, Jose-Andres C., Okenka, Genevieve, Kern, Timothy S., Subauste, Carlos S.
Formato: Texto
Lenguaje:English
Publicado: Molecular Vision 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2713733/
https://www.ncbi.nlm.nih.gov/pubmed/19626134
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author Portillo, Jose-Andres C.
Okenka, Genevieve
Kern, Timothy S.
Subauste, Carlos S.
author_facet Portillo, Jose-Andres C.
Okenka, Genevieve
Kern, Timothy S.
Subauste, Carlos S.
author_sort Portillo, Jose-Andres C.
collection PubMed
description PURPOSE: Advances in the understanding of the pathogenesis of retinal disorders can be facilitated by a methodology to measure expression of proinflammatory molecules in various subsets of retinal cells. METHODS: We examined whether a multiparameter flow cytometric assay can be used to identify various subsets of retinal cells and examine expression of molecules involved in inflammatory responses in the retina. Single-cell suspensions freshly obtained after enzymatic digestion of normal mouse retinas were stained with antibodies against cluster of differentiation 11b (CD11b), cluster of differentiation 31 (CD31), Glial fibrillary acidic protein (GFAP), rhodopsin, Thy-1, and vimentin. These markers were previously shown by immunohistochemistry to label retinal microglia/macrophages, endothelial cells, astrocytes, photoreceptors, ganglion neurons, and Müller cells respectively in normal mouse retinas. RESULTS: Costaining with antibodies against intercellular adhesion molecule-1 (ICAM-1) and CD40 revealed that ICAM-1 is normally expressed at various levels on all subsets of retinal cells examined. In contrast, CD40 was detected only on CD11b(+), CD31(+), Thy-1(+), and vimentin(+) cells. Ischemia-reperfusion of the retina resulted in upregulation of ICAM-1 on CD105(+) and vimentin(+) cells and upregulation of nitric oxide synthase 2 in CD11b(+) cells. DISCUSSION: These results indicate that flow cytometry can be used to readily quantitate expression of surface and intracellular molecules of relevance to retinopathies in freshly isolated retinal cells.
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spelling pubmed-27137332009-07-22 Identification of primary retinal cells and ex vivo detection of proinflammatory molecules using flow cytometry Portillo, Jose-Andres C. Okenka, Genevieve Kern, Timothy S. Subauste, Carlos S. Mol Vis Research Article PURPOSE: Advances in the understanding of the pathogenesis of retinal disorders can be facilitated by a methodology to measure expression of proinflammatory molecules in various subsets of retinal cells. METHODS: We examined whether a multiparameter flow cytometric assay can be used to identify various subsets of retinal cells and examine expression of molecules involved in inflammatory responses in the retina. Single-cell suspensions freshly obtained after enzymatic digestion of normal mouse retinas were stained with antibodies against cluster of differentiation 11b (CD11b), cluster of differentiation 31 (CD31), Glial fibrillary acidic protein (GFAP), rhodopsin, Thy-1, and vimentin. These markers were previously shown by immunohistochemistry to label retinal microglia/macrophages, endothelial cells, astrocytes, photoreceptors, ganglion neurons, and Müller cells respectively in normal mouse retinas. RESULTS: Costaining with antibodies against intercellular adhesion molecule-1 (ICAM-1) and CD40 revealed that ICAM-1 is normally expressed at various levels on all subsets of retinal cells examined. In contrast, CD40 was detected only on CD11b(+), CD31(+), Thy-1(+), and vimentin(+) cells. Ischemia-reperfusion of the retina resulted in upregulation of ICAM-1 on CD105(+) and vimentin(+) cells and upregulation of nitric oxide synthase 2 in CD11b(+) cells. DISCUSSION: These results indicate that flow cytometry can be used to readily quantitate expression of surface and intracellular molecules of relevance to retinopathies in freshly isolated retinal cells. Molecular Vision 2009-07-17 /pmc/articles/PMC2713733/ /pubmed/19626134 Text en http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Portillo, Jose-Andres C.
Okenka, Genevieve
Kern, Timothy S.
Subauste, Carlos S.
Identification of primary retinal cells and ex vivo detection of proinflammatory molecules using flow cytometry
title Identification of primary retinal cells and ex vivo detection of proinflammatory molecules using flow cytometry
title_full Identification of primary retinal cells and ex vivo detection of proinflammatory molecules using flow cytometry
title_fullStr Identification of primary retinal cells and ex vivo detection of proinflammatory molecules using flow cytometry
title_full_unstemmed Identification of primary retinal cells and ex vivo detection of proinflammatory molecules using flow cytometry
title_short Identification of primary retinal cells and ex vivo detection of proinflammatory molecules using flow cytometry
title_sort identification of primary retinal cells and ex vivo detection of proinflammatory molecules using flow cytometry
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2713733/
https://www.ncbi.nlm.nih.gov/pubmed/19626134
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