Anti hla dr alexaflour700

anti-CD14-BV570 (301831, Biolegend), anti-CD11c-PerCP-Cy5.5 (337210), anti-HLA-DR-AlexaFlour700 (307626) all from Biolegend, anti-CD66a/c/e-QDot605 (342302, Biolegend, with in-house conjugation to QDot605, Q-22001), anti-CD16-PE-Cy5 (555408, from BD Biosciences), anti-IL-12-PE (554575), anti-TNF-α-PE-Cy7 (25-7349-82) both from eBioscience, anti-IL-10-PE (506804), anti-IL-6-APC (501112), anti-CD3-BV421 (300434), anti-CD19-BV421 (302233) and anti-CD335-BV421 (331913) all from Biolegend.
Cryopreserved, fixed white cells were washed in PBS and stained for 1 hour at 4°C with surface marker antibodies. For intracellular staining, cells were permeabilized with Perm/Wash buffer (BD Biosciences) and incubated for 1 hour at 4°C with antibodies for intracellular markers. Fixed cells were then acquired on a LSR II flow cytometer (BD Biosciences).

PBMC were isolated from fresh peripheral blood by using density centrifugation (Ficoll-Paque; GE Healthcare). For the analysis of MDSC, PBMC were surface labeled with anti-CD14-BD Horizon V450 (BD Biosciences), anti-HLA-DR-Alexa Flour 700 (Biolegend), anti-CD11b-FITC (eBioscience), anti-CD33-PE-Cy7 (Biolegend), anti-CD15-APC (eBioscience). The gating strategy is depicted in S1 Fig. Expression of CD3ζ was analyzed on PBMC by intracellular staining using a fixation/permeabilization buffer kit (eBioscience) with appropriate mAb anti-CD3ζ-PE (Exbio). For the analysis of IFN-γ and IL-17A producing cells, PBMC were stimulated with PMA (50 ng/ml; Sigma-Aldrich) plus ionomycin (1 mg/ml; Sigma-Aldrich) for 4h in the presence of brefeldin A (5 mg/ml; Biolegend). Intracellular IFN-γ and IL-17A staining were assessed using a fixation/permeabilization buffer kit (eBioscience) with appropriate mAb IFN-γ-Pacific Blue (Biolegend) and anti-IL-17-A647 (Biolegend) and measured by flow cytometry. Data were acquired by LSRFortessa and LSR II flow cytometers (BD Biosciences) and analyzed using FlowJo software (Tree Star).