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Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37°C with PMA/...

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Autores principales: Foerster, Anna, Assenmacher, Mario, Niemoeller, Michaela, Rankin, Elly, Mohaupt, Mariette, Richter, Anne
Formato: Texto
Lenguaje:English
Publicado: MyJove Corporation 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2865976/
https://www.ncbi.nlm.nih.gov/pubmed/19229172
http://dx.doi.org/10.3791/1037
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author Foerster, Anna
Assenmacher, Mario
Niemoeller, Michaela
Rankin, Elly
Mohaupt, Mariette
Richter, Anne
author_facet Foerster, Anna
Assenmacher, Mario
Niemoeller, Michaela
Rankin, Elly
Mohaupt, Mariette
Richter, Anne
author_sort Foerster, Anna
collection PubMed
description The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37°C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37°C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.
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spelling pubmed-28659762011-03-14 Detection and Isolation of Viable Mouse IL-17-Secreting T Cells Foerster, Anna Assenmacher, Mario Niemoeller, Michaela Rankin, Elly Mohaupt, Mariette Richter, Anne J Vis Exp Immunology The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37°C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37°C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads. MyJove Corporation 2008-12-18 /pmc/articles/PMC2865976/ /pubmed/19229172 http://dx.doi.org/10.3791/1037 Text en Copyright © 2008, Journal of Visualized Experiments http://creativecommons.org/licenses/by/2.0/ 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 Immunology
Foerster, Anna
Assenmacher, Mario
Niemoeller, Michaela
Rankin, Elly
Mohaupt, Mariette
Richter, Anne
Detection and Isolation of Viable Mouse IL-17-Secreting T Cells
title Detection and Isolation of Viable Mouse IL-17-Secreting T Cells
title_full Detection and Isolation of Viable Mouse IL-17-Secreting T Cells
title_fullStr Detection and Isolation of Viable Mouse IL-17-Secreting T Cells
title_full_unstemmed Detection and Isolation of Viable Mouse IL-17-Secreting T Cells
title_short Detection and Isolation of Viable Mouse IL-17-Secreting T Cells
title_sort detection and isolation of viable mouse il-17-secreting t cells
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2865976/
https://www.ncbi.nlm.nih.gov/pubmed/19229172
http://dx.doi.org/10.3791/1037
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