Ifng fitc

Antibodies for intracellular cytokine staining included: anti-CD3-AF700, CD4-PcPCy5.5, CD8-PECF594, IFNg-FITC, CD14-V500, CD19-V500, TNFα-APC, CD38-PECy7, HLADR-BV605 (BD Biosciences); PD-1-PE, IL-2-BV421 (BioLegend); Live-Dead-AquaViD (LifeTechnologies).
The T regulatory cell/activation panel included: anti-CD3-AF700, CD25-PE, HLA-DR-FITC (BD); CD4-PETxR, CD8-APC AF750, Live-Dead-AquaViD (LifeTechnologies); CD39-PECy7, FOXP3-APC (eBioscience); CD127- PE Cy5.5 (Beckman Coulter); CD38- BV421 (BioLegend).

PBMCs were cultured for 96h under different experimental conditions. Cells were counted, aliquoted and directly stained with 20μl (without fixation and permeabilization, as indicated by the manufacturer) with a CD8/CD69/CD3 antibody cocktail (BD FastImmune, BD Biosciences, cat# 340367) for analysis of activation. For immunophenotyping of the Th1/Th2/Th17 response, a different aliquot of cells was washed, fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) and stained with CD4-Percp-Cy5.5, 20μl test (cat# 341654), IFN-g-FITC, 5 μl/sample (cat# 554700), IL-4-PE-Cy7, 5 μl/sample (cat# 560672) and IL-17A- PE, 5 μl/sample (cat# 560486). Tregs were identified in a third aliquot of PBMCs that was washed, fixed and permeabilized with Human FOXP3 Buffer Set (BD Biosciences - cat# 560098), and stained with CD4-Percp-Cy5.5, 20 μl/sample (cat# 341654), FoxP3-AlexaFluor 488, 20 μl/sample (cat# 560047) and CD25-PE, 20 μl/sample (cat# 555432; all BD Biosciences). A minimum of 10,000 events were acquired in a FACSVerse flow cytometer and analyzed with FacSuite software (BD Biosciences).