Cells were blocked with normal sheep serum and stained with monoclonal anti-mouse CD4 antibodies (RM4-5 clone; eBiosciences; San Diego, CA, USA). Intracellular staining of CD4 T cells was performed using anti-mouse IL-10-PE antibody (JES5-16E3 clone), anti-IL-4 PECy7 (11B11 clone), and anti-IL-17 eFluor 660 (eBio17B7 clone) or the respective controls IgG2bκ PE, IgG1κ PECy7, and IgG2aκ eFluor 660 (all against mouse proteins, from eBioscience). Treg cells were characterized by staining for CD4, CD25, and Foxp3, according to the manufacturer's instructions (Mouse Treg Staining Kit, eBiosciences).
Pulmonary myeloid dendritic cells (CD11c+CD11b+Gr1−B220−) were characterized as previously [26 (link)], using anti-Gr1 FITC (RB6-8C5 clone) anti-CD11c PE (N418 clone), anti-CD11b PEcy7 (M1/70 clone), and anti-B220 APC (RA3-6B2 clone) or the respective controls IgG2bκ FITC, IgG armenian hamster PE, IgG2bκ PECy7, and IgG2aκ APC (all against mouse proteins, from eBiosciences).
All data were acquired in a FACSCalibur flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA, USA) and analyzed using the FlowJo X 10 and 7.6.5 software (Tree Star Inc., Ashland, OR, USA). Percentages of total cells were presented in bar graphs.
Pulmonary myeloid dendritic cells (CD11c+CD11b+Gr1−B220−) were characterized as previously [26 (link)], using anti-Gr1 FITC (RB6-8C5 clone) anti-CD11c PE (N418 clone), anti-CD11b PEcy7 (M1/70 clone), and anti-B220 APC (RA3-6B2 clone) or the respective controls IgG2bκ FITC, IgG armenian hamster PE, IgG2bκ PECy7, and IgG2aκ APC (all against mouse proteins, from eBiosciences).
All data were acquired in a FACSCalibur flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA, USA) and analyzed using the FlowJo X 10 and 7.6.5 software (Tree Star Inc., Ashland, OR, USA). Percentages of total cells were presented in bar graphs.
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