Cd86 fitc

Antibodies for surface markers staining of DCs and T cells were obtained from BD Biosciences, New York, USA (anti-human CD3-PE, CD3-FITC, CD8-PerCP, CD8-APC, CD56-PE, NKG2D-APC, CD4-FITC, CD4-PerCP, CD107a-FITC, CD25-APC, CD45RO-FITC, CD27-PerCPCY5.5, CD57-APC, CCR7-PE, CD14-APC, CD80-PE, CD83-APC, CD86-FITC, HLA-DR-FITC). Antibodies for intracellular proteins staining were also obtained from BD Biosciences (anti-human IFNγ-APC, TNFα-PECY7, granzyme B-FITC, FoxP3-PE). The intracellular staining was performed by fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences). The experiments were performed by using FACS CantoII (BD Biosciences) flow cytometer, and data were analyzed by using the Flowjo software.

Cells were stained with specific antibodies or control isotypes using conventional protocols. The following anti-mouse antibodies were used for FACS analysis of MDSCs: CD11b/PECy5, Gr1/PE, Gr1/APC, CD62L/FITC, F4/80/FITC, CD11c/PE, and MHC CI OVA peptide/Biotin from eBiosciences (San Diego, CA); as well as CD124/PE, CD40/FITC, CD86/FITC, I^A-I^E/FITC, Ly6C/FITC and Ly6G/PE that were purchased from BD Biosciences (San Jose, CA). Lymphocytes characterization was performed with the next specific antibodies: CD8/PE and CD8/Biotin, CD69/PECy5, CD62L/FITC, CD247/PE, CD4/PE, CD4/PECy5, CD25/PECy5, Foxp3/Biotin, IFN-γ/FITC, IL-4/PE and IL-17/PE, all from eBiosciences. FITC-conjugated Streptavidin was purchased also from eBiosciences. In all cases, cells were previously incubated with anti-mouse FcγR 2.4G2 ascites (HB-197; ATCC) to reduce the non-specific binding. For intracellular staining, cells were fixed and permeabilized, according to the manufacturer’s protocol, with either Foxp3 Staining Buffer Set or IC Fixation and 10X Permeabilization Buffers from eBiosciences. Cells were acquired using both FACScan (BD Biosciences) and Gallios (Beckman Coulter, Miami, FL) flow cytometers and analyzed with Kaluza 1.2 (Beckman Coulter) and FlowJo 5.7.2 (Tree Star Inc., USA) softwares.

Cells were fixed with paraformaldehyde (PFA) and treated with either BSA (extracellular staining) or BSA/Saponin to measure intracellular staining. The following antibodies were used to detect Zika virus: anti-Flavivirus 4g2 mouse IgG2a (NovusBio), anti-Flavivirus 4g2 monoclonal rabbit (Absolute Antibody), anti-Zika virus monoclonal Rabbit (Genetex).
All other antibodies were anti-human: DC-SIGN mouse IgG1 (AZN-D1), anti-langerin mouse IgG1 (10E2) both in house made, PE conjugated CD207 (langerin), APC conjugated CD1a (BD Biosciences), CD86-FITC (BD Pharmingen), CD80-PE (BD Pharmingen), CD83-APC (BD Pharmingen), DC-SIGN-FITC (R&D systems), CD3-APC/Fire750 (Biolegend), CD11c-APC (Biolegend), PEcy7-HLA-DR (BD Pharmingen), APCcy7-CD14 (BD Biosciences), APCcy7-CD11c (Biolegend),APC-AXL (Thermofisher, PE-MerTK (Thermofisher) and anti-Tyro3 (Thermofisher). For secondary detection the following antibodies were used: AF488-conjugated goat anti-mouse IgG2a (Invitrogen), AF647-conjugated donkey anti-rabbit (Thermofisher), FITC-conjugated goat-anti-mouse IgM (Invitrogen), AF488-conjugated donkey anti-rabbit (Thermofisher).
Flow cytometric analyses were performed on a BD FACS Canto II (BD Biosciences) and data was analysed using FlowJo V10 software (TreeStar).

Following Fc block (eBiosciences, 93) cells were stained with the following antibodies: CD11a-PE (BioLegend, 2D7), CD18-FITC (BD Biosciences, C71/16) CD11c-PE-Cy7 (eBioscience, N418), CD11b-APC (BioLegend, M1/70), CD80-APC (eBioscience, 16-10A1), CCR7-PE (BioLegend, 4B12), CD40-PE (BioLegend, 3/23), CD86-FITC (BD Biosciences, GL1). FITC-conjugated antibodies were used at 1:100 dilution, all other antibodies were used at 1:200 dilution. Data were acquired using a LSRForetessa (BD) and analyzed using FlowJo software (TreeStar, USA).

Expressions of cell surface co-stimulatory molecules (CD80 and CD86) are markers of DC maturation.^{53 (link)} We assessed CD maturation and activation in EC cells. In brief, EC cells were treated with RT (6 Gy), ERAD inhibitors (EerI, 5000 nM; NMS-873, 500 nM), and the combination therapy, and maintained for 24 hours. Then, these cells were washed and cocultured with immature DCs at a ratio of 1:1 for at 37°C for 24 hours. To confirm the maturation of DCs, the harvested cells were stained with markers of maturation and activation (CD80-FITC, CD86-FITC, CD11c-PE; BD Biosciences, USA). The labeled cells were analyzed by flow cytometry.

Monoclonal antibodies against human CD80-PE, CD83-FITC, CD86-FITC, CD40-FITC, CD38-PE, CD74-FITC (BD Biosciences), and CCR7-FITC (R&D Systems, Minneapolis, MN, USA) were used to detect surface markers on DCs using flow cytometry. Cell debris was eliminated by forward and side scatter gating. The data were analyzed with WinMDI, version 2.9 (Bio-Soft Net).