Lymphocyte function analysis
FLUOR
Using Beckman Coulter’s custom design service and its dry coating technology, custom tubes containing anti-IFN-γ-FITC (clone 45.15), anti-CD25-PE (clone B1.49.9), anti-CD4-PE-Cy5.5 (clone 13B8.2), anti-IL-4-PE-Cy7 (clone MP4-25D2), anti-Foxp3-Alexa Fluor 647 (clone 259D), anti-TNF-α-Alexa Fluor 700 (clone IPM2), anti-CD3-APC-Alexa Fluor 750 (clone UCHT1), anti-IL-17A-PacificBlue (clone BL168) and anti-CD8-KromeOrange (clone B9.11) were produced. Liquid antibody was also used: anti-IL-2-BV605 (BioLegend, clone MQ1-17H12).Staining procedure: 100 μl of total heparinized blood was added to a DURActive 1 tube containing phorbol-myristate-acetate, ionomycin and brefledin A (C11101, Beckman Coulter) for 3 h at 37 °C in the dark. After activation, 25 μl of PerFix-NC R1 buffer (PerFix-NC Kit, B31168, Beckman Coulter) was added on vortex and incubated for 15 min at room temperature. Then, 2 ml of PBS 1× was added, and, after centrifugation, the pellet was resuspended in 25 μl of FBS (Dutscher), and 300 μl of PerFix-NC R2 buffer was added. A 325-μl aliquot was transferred to a DURAClone tube containing the liquid antibody, vortexed immediately for 15 s and incubated for 1 h at room temperature in the dark. PBS 1× (3 ml) was added to the tubes and incubated for 5 min at room temperature in the dark before centrifugation for 5 min at 500After the acquisition, validation of the compensations was performed for each .fcs file on Kaluza analysis software (Beckman Coulter), and then an unsupervised analysis was performed.