During the last 6 h of culture, brefeldin A (1 µg/mL) (eBioscience) was added to improve cytokine detection. The cells were then washed and stained for surface markers, and fixed using 2% formaldehyde (Sigma-Aldrich). Fixed cells were permeabilized and stained using anticytokine monoclonal antibodies. Immunoglobulin control antibodies and a control of unstimulated PBMC were included in all experiments [45] (link).
Monoclonal antibodies directly conjugated with fluorocromes were: anti-CD4 APC-Cy7 (BD Biosciences), CD25 PE-Cy7 (eBioscience), CD14 PerCP (eBioscience), Foxp3 Alexa fluor 488 (BD Biosciences), TGF- β1 PerCP (R & D Systems), IL-10 APC (BD Biosciences) and IL-17F PE (eBioscience).
Preparations were acquired on BD FACSCanto II (BD Biosciences) and 30,000 events were acquired for the analysis. Data was processed using FlowJo software, version 7.6.5 (Tree Star Inc, Ashland, OR, USA). Specific gating strategies to select Treg CD4+CD25highFoxp3+, CD4+CD25−Foxp3− and monocytes (CD14+) subsets are presented through representative density plots which are shown inFigure 1 . Treg cells were stained and analyzed for TGF-β1, IL-10 and IL-17, while monocytes were stained for IL-10.
Monoclonal antibodies directly conjugated with fluorocromes were: anti-CD4 APC-Cy7 (BD Biosciences), CD25 PE-Cy7 (eBioscience), CD14 PerCP (eBioscience), Foxp3 Alexa fluor 488 (BD Biosciences), TGF- β1 PerCP (R & D Systems), IL-10 APC (BD Biosciences) and IL-17F PE (eBioscience).
Preparations were acquired on BD FACSCanto II (BD Biosciences) and 30,000 events were acquired for the analysis. Data was processed using FlowJo software, version 7.6.5 (Tree Star Inc, Ashland, OR, USA). Specific gating strategies to select Treg CD4+CD25highFoxp3+, CD4+CD25−Foxp3− and monocytes (CD14+) subsets are presented through representative density plots which are shown in