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

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°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 bispecific 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. Video LinkThe video component of this article can be found at https://www.jove.com/video/1037/ ProtocolPreparing the Reagents 1. Make a buffer containing PBS with BSA (0.5%), and 2 mM EDTA. Because air bubbles can block MACS separation columns, the buffer needs to be degassed and stored at 2-8°C before use. 2. We use RPMI 1640 medium containing 5% mouse serum. The culture medium should not contain BSA or FCS, as these compounds will alter the specificity of cell stimulation. 3. The Mouse IL-17 Secretion Assay -Cell Enrichment and Detection Kit from Miltenyi-Biotec. The kit contains the following components: the IL-17 Catch Reagent, the IL-17 Detection Antibody (Biotin), the Anti-Biotin-PE, and the Anti-PE MicroBeads. Stimulating the Splenocytes1. This protocol is performed in the presence of a negative and positive control, such as unstimulated splenocytes and a counterstain for T cells. This protocol is carried out using sterile technique. 2. Prepare a single cell suspension of mouse splenocytes that were isolated using the gentleMACS™ Dissociator. The concentration of cells should be predetermined via cell counting. Pellet the cells at 200×g for 10 minutes at room temperature. 3. Following centrifugation, aspirate the supernatant off the pellet using a pipette. Do not decant the tube to avoid loss of the pellet. 4. Now, resuspend the cells in the culture medium and then add to a well. Add sufficient medium for a concentration of ten million cells per mL and five million cells per square cm. 5. To stimulate an immune response in our resuspended cells, we add ionomycin (1 μg/mL) and PMA (10 ng/mL) to the sample and mix the solution by gently pipetting up and down. Then the wells are labeled accordingly. 6. Now, we will incubate our cells for 3 hours at 37 °C with no mixing to start the stimulation period. Proceed to IL-17 analysis 3 hours from the onset of stimulation, so plan accordingly. 7. To stop secretion, cells are placed on ice and we collect the stimulated cells by gently pipetting up ...