*4.7. Cytokines Serum Levels*

Samples were obtained in sterile clotting tubes from peripheral blood by ante-cubital venipuncture. These samples were centrifuged at 863× *g* for 20 min at 4 ◦C, aliquoted, identified and labeled, and frozen at –80 ◦C. Then, they were thawed and evaluated using the high sensitivity human MILLIPLEX® kit to simultaneously measure IFNγ, IL-4 and IL-17A (Millipore) following the manufacturer's instructions and revealing the results by Luminex (MAGPIX® system). The tested cytokines had the following

sensitivity limits (0.48 pg/mL for IFNγ, 1.12 pg/mL for IL-4 and 0.33 pg/mL for IL-17A). The results were analyzed using Analyst 5.1 software MILLIPLEX® (MerkMillipore, Boston, MA, USA).
