Cytofix cytoperm and perm wash

Cell suspensions were incubated with fluorochrome labeled-Abs for 30 min at 4°C. Different combinations of the following anti-mouse Abs (BD Biosciences, eBioscience, Biolegend) were used: PerCP-Cy5.5-conjugated anti-CD4, FITC-conjugated anti-CD8 and anti-CD3, PE-conjugated anti-CD25, APCconjugated anti-gd-TCR and anti-CD19, PE-Cy7-conjugated anti-NK1.1, APC-Cy7-conjugated anti-B220, PE-conjugated anti-PD-1, anti-PD-L1 and anti-PD-L2. Transcription factors Forhead box P3 (FoxP3) and interferon-regulatory factor-4 (IRF-4) were detected after cell fixation and permeabilization with FoxP3 Staining Buffer Set (eBioscience) using the following antibodies: APC-conjugated anti-FoxP3 and eFluor660conjugated anti-IRF4 (eBioscience). For intracellular cytokine staining, DCs or allogenic cultures were exposed to brefeldin A (10 μg/ml; Sigma) for the last 4 h of cell culture. Cells were fixed and permeabilized with Cytofix/Cytoperm and Perm/Wash (BD Biosciences) and incubated with surface staining antibodies and PE-conjugated anti-IL-17 and APC-conjugated anti-IFN-g (Biolegend). Cells were acquired on BD FACSCanto II and analyzed using the FlowJo software.