Anti-mouse (CD3, CD11b, CD19, IgM, CD49b, Ter119) PE or CD11b PE-CY7, CD11c FITC or APC-CY7, B220 PERCP-CY5.5 and PDCA1 ef450 were used for pDC analysis and purchased from BD Bioscience (San Jose, CA), Biolegend (San Diego, CA), and eBioscience (San Diego, CA). Stimulation of pDC for cytokine profile analysis was done using 50μM of CpG (ODN 1585, Invivogen, San Diego, CA) of whole bone marrow in complete RPMI 1640 supplemented with 10% FBS, 100 U/mL of penicillin, 100μg/mL of streptomycin, and 50μM each of 2-mercaptoethonaol, nonessential amino acids, HEPES, and sodium pyruvate (complete media) in 10cm wells for 9 hours at 37°C. BD golgiplug was added at hour 3. Intracellular analysis of pDC was done using BD Bioscience cytofix/cytoperm kit and anti-mouse IDO PerCP-Cy5.5, IFNα FITC, IL-10 PECY7 and IL-12 APC antibodies.
Splenocytes were stained using anti-mouse CD3 FITC, CD4 PE-CF594, CD8 PERCP-CY5.5, and CD25 APC-CY7. T cells were stimulated with BD leukocyte activation cocktail and golgiplug for 6 hours. Intranuclear staining was done using the eBioscience fixation kit and Tbet PE-CY7, GATA3 PE, RORγT APC, and FoxP3 PE antibodies. Data were acquired with a FACS Aria (BD, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland, Oregon).
Splenocytes were stained using anti-mouse CD3 FITC, CD4 PE-CF594, CD8 PERCP-CY5.5, and CD25 APC-CY7. T cells were stimulated with BD leukocyte activation cocktail and golgiplug for 6 hours. Intranuclear staining was done using the eBioscience fixation kit and Tbet PE-CY7, GATA3 PE, RORγT APC, and FoxP3 PE antibodies. Data were acquired with a FACS Aria (BD, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland, Oregon).