Il 17a fitc

T-lymphocytes were analyzed in PBMCs by nine-color flow-cytometry. PBMCs were incubated with the next surface-labeled monoclonal-antibodies, CD3-PercP, CCR7-PECY7 (Becton-Dickinson, San José, CA, USA), CD8-Alexa405, CD45RA-APC (Caltag, San Francisco, CA, USA) and CD27-APCAlexa780 (eBioscience, San Diego, CA, USA).
For intracytoplasmic staining, PBMCs were fixed and permeabilized (Fix and Perm, Caltag, San Francisco CA, USA), and cytokines were stained with IL-4-PE, IFNγ Alexa700 and IL-17A-FITC (Becton-Dickinson, San José, CA, USA). All samples were stained with a dead cell-discriminator simultaneously with antibody addition (fixable aqua dead cell stain kit for 405 nm excitation; Molecular Probes, Eugene, OR). Samples were acquired in a FacsAria-II flow-cytometer and were analyzed using FacsDiva 5.0 and Flow-Jo 10.0 software.

T-lymphocytes were studied in PBMCs by nine-color flow cytometry. PBMCs were incubated with the next surface-labeled monoclonal-antibodies, CD3-PercP, CCR7-PECY7 (Becton-Dickinson, BD, CA, USA), CD8-Alexa405, CD45RA-APC (Caltag, Carlsbad, CA, USA) and CD27-APCAlexa780 (eBioscience, San Diego, CA, USA).
For intracytoplasmic staining, cells were fixed and permeabilized (Fix and Perm, Caltag, Carlsbad, CA, USA), and cytokines were stained with IL-4-PE, IFNγ-Alexa700 and IL-17A-FITC (Becton-Dickinson, BD, CA, USA). All samples were stained with a dead cell-discriminator simultaneously with antibody addition (Fixable aqua dead cell stain kit for 405 nm excitation; Molecular Probes, Eugene, OR, USA).
Samples were acquired in a FacsAria-II flow cytometer and were analyzed using FacsDiva 5.0 and Flow-Jo 7.0 software (Becton-Dickinson, BD, San Jose, CA, USA).