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 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.
Foerster
Anna
A
Miltenyi Biotec, GmbH.
Assenmacher
Mario
M
Niemoeller
Michaela
M
Rankin
Elly
E
Mohaupt
Mariette
M
Richter
Anne
A
D000818:Animals; D005434:Flow Cytometry / Q000379:methods; D020381:Interleukin-17 / Q000032:analysis* / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D051379:Mice; D012636:Secretory Rate / Q000187:drug effects; D013154:Spleen / Q000166:cytology / Q000187:drug effects; D013194:Staining and Labeling / Q000379:methods; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D020381:Interleukin-17; D015759:Ionomycin; D013755:Tetradecanoylphorbol Acetate