Flow Cytometry

Flow cytometric analysis of T-cell subpopulations was performed on a MACSQuant^® device (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Samples were prepared as follows: within 15 min after blood draw, EDTA-treated blood was added to Schwinzer red blood cell lysis solution. After 15 min of incubation at 4°C the cells were washed and buffered with PBA and plated on 96-well round bottom plates for staining. Fc-blocking agent was added to prevent unspecific antibody binding.

After incubation we used APC-labeled anti-CD4, eFluor450™-labeled anti-CD161, and FITC-labeled anti-CD196 for detection of Th17 cells. For CD4+ Treg detection we used FITC-labeled anti-CD4, eFluor450™-labeled anti-CD25, and APC-Cy7-labeled anti-CD127. Cell activation was evaluated using PE-labeled anti-IL17A. After incubation, cells were washed and resuspended in PBA buffer for immediate flow cytometric analysis. Prior to measurements, the MACSQuant^® was calibrated using MACSQuant^® calibration beads according to the manufacturer's instructions.

The obtained data sets were analyzed using FlowJo Software (FlowJo LLC, Ashland, OR). After single cell selection, Th17 cells were defined as CD4+, CD161+, CD196+ cells, and CD4+ Tregs were defined as CD4+, CD25+, CD127⁻ cells. IL-17A expression on Th17 cells was assessed by calculating the relative median fluorescence intensity (MFI) of PE on Th17 cells stained with PE-conjugated anti-IL17A antibody. To calculate the relative MFI, the MFI of PE on anti-IL17A stained cells was divided by the MFI of PE measured on Th17 cells not stained with any PE-conjugated antibody. IL-17A expression on CD4+ Tregs was assessed analogously.