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Detection and Isolation of Viable Mouse IL-17-Secreting T Cells
Published on: December 18, 2008
Detection and isolation of viable mouse IL-17-secreting T cells
Anna Foerster1, Mario Assenmacher, Michaela Niemoeller
1Miltenyi Biotec, GmbH.
Insights
This study details a method to detect and isolate interleukin-17 (IL-17)-secreting cells using MACS Cytokine Secretion Assay technology. This technique enables sensitive identification and isolation of viable cytokine-producing immune cells.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Cytokine secretion assays are crucial for understanding immune responses.
- Identifying specific cytokine-producing cells, like those secreting IL-17, is vital for immunological research.
- Existing methods may lack sensitivity or the ability to isolate viable cells.
Purpose of the Study:
- To present the MACS Cytokine Secretion Assay technology for detecting and isolating IL-17-secreting cells.
- To demonstrate a method for sensitive, single-cell level analysis of cytokine secretion.
- To enable the isolation of viable cytokine-secreting cells for further study.
Main Methods:
- Mouse splenocytes were stimulated with PMA/ionomycin to induce cytokine secretion.
- The IL-17 Catch Reagent, a bispecific antibody, was used to capture secreted IL-17 near the cell surface.
- Cells were analyzed via flow cytometry or prepared for isolation using Anti-PE conjugated MicroBeads after antibody incubation.
Main Results:
- The assay successfully labels IL-17-secreting cells.
- The method allows for the detection of secreted IL-17 at the single-cell level.
- Viable cytokine-secreting cells can be isolated and enriched using this technology.
Conclusions:
- The MACS Cytokine Secretion Assay provides a sensitive method for detecting and isolating IL-17-secreting cells.
- This technology facilitates the study of immune responses by enabling the analysis and isolation of specific cytokine-producing cells.
- The assay is valuable for immunological research requiring single-cell resolution and cell isolation.
Abstract:
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 degrees 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 degrees 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.
