Anti pdl 1 biotin

Antibodies were purchased as followed: Anti-CD4-V450, anti-CD8α-APCcy7, anti-B220-V450, anti-CD138-Pe-cy7, anti-CD86-Pe-cy5, anti-FAS-PE, anti-GL-7-APC, anti-CD40-APC, anti-MHCII-FITC, anti-IL-4-PE, anti-IL-5-PE, anti-IgM-Pe-cy7, anti-IgG1-APC, anti-IFN-r-Percp5.5, anti-TNF-α-PE, and anti-IL-17-Pe-cy7 were purchased from BD Biosciences (Franklin Lakes, NJ). Anti-PDL-1-biotin (eBioscience, San Diego, CA), anti-XBP-1s (Cell Signaling Technology, Danvers, MA), and anti-Rabbit IgG-FITC (Thermo Fisher Scientific, Waltham, MA) antibodies were purchased from commercial sources. Recombinant mouse IL-4 (PeproTech, Rocky Hill, NJ) and LPS (Sigma-Aldrich, St. Louis, MO) were purchased from the commercial companies. Goat F(ab’)2 Anti-Mouse IgM (1022-01, SouthernBiotech, Birmingham, AL) and anti-mouse CD40 (BE0016-2, BioXCell, Lebanon, NH) were commercially purchased from companies.

For immunophenotyping, activated DCs or tolerogenic DCs were incubated with monoclonal antibody (mAb)- fluorochrome or biotin conjugates: anti-CCR7-PerCP, anti-CD11c-FITC, anti-CD11b-PE, anti-CD40-PE, anti-CD80-PE, anti-CD86-PE, anti-MHC-II-biotin, and anti-PD-L1-biotin (eBioscience Inc.; San Jose, CA) or with the corresponding isotype controls for 20 min at 4°C in the dark and washed twice with saline solution with 1% FBS. PE-avidin was added to the cell suspensions previously incubated with biotin-mAb conjugates, for 20 min at 4°C in the dark, followed by washing once with 1% FBS saline solution. For each sample, data from 100,000 cells was acquired by three-color flow cytometry, using a BD LSRFortessa SORP cytometer and a BD FacsDiva v.6.2 software (Becton Dickinson; Heidelberg, Germany). Cell-free supernatants of mDCs or tDCs were collected 24 h after stimulation and stocked at −20°C until used for cytokine measurements. The concentrations of IL-6, IL-10 and IL-12 cytokines were measured by ELISA, using specific antibody kits (R&D Systems, Minneapolis, MN), according to manufacturer's instructions.