Pe conjugated anti pd 1

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized blood by Ficoll density gradient centrifugation. For phenotypic analysis, the cells were washed twice and stained for 30 min on ice with mixtures of fluorescence-conjugated surface mAbs or isotype-matched controls, and the cells were then washed twice and resuspended in PBS buffer for flow cytometry analysis. PBMCs were stained with the following antibodies: phycoertthrin-cyanin 5 (PE-Cy5)-conjugated anti-CD3 (eBioscience, San Diego, CA, USA); fluorescein isothiocyanate (FITC)-conjugated anti-CD4, anti-CD8 and anti-CD19 (eBioscience); phycoertthrin-cyanin7 (PE-Cy7)-conjugated anti-CD4 (eBioscience); PE-conjugated anti-PD-1 (eBioscience) and anti-IFN-γ (eBioscience).

Cell suspensions were prepared from spleen, lymph nodes, VAT, or liver of ob/ob mice as previously described (Chang et al., 2015 (link); Xiang et al., 2016 (link)). DC single-cell suspensions were obtained from bone marrow following standard procedures. Cells were stained with antibodies against the following cell surface antigens: fluorescein isothiocyanate (FITC)-conjugated anti-CD86, allophycocyanin (APC)-conjugated anti-CD11c, phycoerythrin (PE)-conjugated anti-MHC-II and PE-conjugated anti-PD-L1 (eBioscience, USA); APC-conjugated anti-Foxp3, FITC-conjugated anti-CD4, PE-conjugated anti-CD25 (eBioscience, USA); APC-conjugated anti-CD3, PE-conjugated anti-PD-1, and PE-conjugated anti-TCRβ (eBioscience, USA). Samples were detected on a BD LSRII flow cytometer or BD C6 (BD Pharmingen), and data were analyzed with Flowjo software, version 10.1.

Fluorescently conjugated antibodies were purchased from BD Biosciences, eBioscience, TONBO biosciences, and Bioss for phenotypic analyses. The following panel of mouse anti-human mAbs was used: APC.cy7-conjugated anti-CD3 (BD Biosciences, 557832, SK7) was purchased from BD Biosciences (San Jose, CA, USA), Percp.cy5.5-conjugated anti-CD4 (TONBO biosciences, 65-0048-T100, OKT4) was purchased from TONBO biosciences, Inc. (San Diego, CA, USA), PE-conjugated anti-PD-1 (eBioscience,12-2799-42, eBioJ105) was purchased from eBioscience, Inc. (San Diego, CA, USA), and AF-488-conjugated-anti-AR (Bioss, bs-0118R-AF488) and APC-conjugated-ER (Bioss, bs-0174R-APC) were both purchased from Bioss Antibodies Inc. (Woburn, Massachusetts, USA). Estradiol valerate (Selleckchem, S4757) and dihydrotestosterone (Selleckchem, S3149) were all purchased from Selleckchem (Houston, TX, USA). The PBMCs from healthy donors and lung cancer patients were stained for flow cytometry. The cells were then harvested and stained for flow cytometric analysis. All samples were collected on FACSAria II BD (Mountain View, CA, USA). Data were analyzed using Flow Jo software (Tree Star, San Carlos, CA, USA).