Immature DCs were generated as described above (“Generation of Mo-DCs” section). DCs were cultured alone or with untreated or BTZ pretreated-AMO1 (5nM) for 24h. DCs alone were cultured i) without maturation stimuli; ii) with 50 ng/ml of TNFalpha (Millipore Sigma); or iii) with 5nM of BTZ. After 24h, cells were harvested and analyzed by flow cytometry using the following Abs: anti- CD83-APC (#551073), CD86-FITC (#555657) and 7-AAD from BD Biosciences and CD11c-BV650 (#563404, Biolegend). Dead cells were excluded by 7-AAD positivity, and CD83 and CD86 expression was evaluated on CD11c+ cells.
Anti cd83 apc
Manufactured by BD
Sourced in United States
Anti-CD83-APC is a monoclonal antibody conjugated with allophycocyanin (APC) that targets the CD83 cell surface antigen. CD83 is a type I transmembrane glycoprotein that is expressed on mature dendritic cells and is involved in the regulation of the immune response. The Anti-CD83-APC conjugate can be used for the identification and analysis of CD83-positive cells in flow cytometry applications.
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Lab products found in correlation
Anti cd80 pe, by BD (4 mentions)
Anti cd86 fitc, by BD (2 mentions)
Anti cd80 fitc, by Beckman Coulter (2 mentions)
Anti cd69 pc5, by Beckman Coulter (2 mentions)
Anti cd86 pc5, by Beckman Coulter (2 mentions)
Anti cd197 apc cy7 ccr7, by BioLegend (2 mentions)
Anti cd8 pacific blue, by BioLegend (2 mentions)
Anti cd14 pc7, by Beckman Coulter (2 mentions)
Anti hla dr ecd, by Beckman Coulter (2 mentions)
Anti cd1a pe, by Beckman Coulter (2 mentions)
Kaluza software, by Beckman Coulter (2 mentions)
Anti cd4 fitc, by Beckman Coulter (2 mentions)
Anti hla dr percp, by BD (2 mentions)
Anti cd3 pe, by BD (2 mentions)
Cd11c bv650, by BioLegend (1 mentions)
Cd86 fitc, by BD (1 mentions)
Tnfalpha, by Merck Group (1 mentions)
7 aad, by BD (1 mentions)
Anti cd83 fitc, by BD (1 mentions)
Anti cd40 pe, by Beckman Coulter (1 mentions)
9 protocols using anti cd83 apc
1
Immature DC Maturation Assay
2
Analyzing Dendritic Cell Activation
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CD1c+ DCs and pDCs were incubated overnight at 37°C with different stimuli in a 96-well round-bottom plate, either separate or together at a 1:1 ratio, with each well containing equal cell numbers (50 × 103 cells in 100 μL). After overnight culture, supernatants were taken and cells were stained with the following primary monoclonal antibodies: anti-human leukocyte antigen (HLA)-ABC-APC (BD Biosciences, 555555), anti-HLA-DR/DP/DQ-FITC (BD Biosciences, 555558), anti-CD80-PE (BD Biosciences, 557227), anti-CD83-FITC (BD Biosciences, 556910), anti-CD83-APC (BD Biosciences, 551073), anti-CD86-PE (BD Biosciences, 555658), anti-PDL-1-PE (BD Biosciences, 557924), anti-CD40-PE (Beckman Coulter, PN IM1936U), anti-CCR7-PE (Miltenyi Biotec, 130-093-621). Anti-CD11c-PE (BD Biosciences, 333149) or anti-CD123-APC (Miltenyi Biotec, 130-090-901) primary monoclonal antibodies were used to distinguish between DC subsets in co-cultures. Samples were measured on a FACSCalibur or FACSVerse (BD Biosciences) and analyzed by FlowJo software (TreeStar, Inc.). The results are depicted as geometric mean fluorescence intensity (MFI) normalized to the negative control. Supernatants were analyzed for IL-12p70 (Thermo Fisher Scientific, M122), TNF-α (eBioscience, 88-7346-88) and IFN-α (Bender Medsystems, BMS216MST) by sandwich ELISA.
3
HSV-CD80 Infected Dendritic Cell Analysis
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BM-derived DCs were infected with 1 PFU/cell of HSV-CD80 and parental virus or mock infected for 24 hr. Infected or mock-infected cells were harvested and stained with anti-CD11c-PacBlue, anti-CD80-PE, anti-PD-L1-APC, anti-HSV-1 gC-FITC, anti-CD83-APC, and anti-CD69-PE from BD PharMingen (San Diego, CA) and Biolegend (San Diego, CA) and then analyzed by FACS as previously described [69] (link).
4
Phenotypic Characterization of Murine Splenocytes and Neutrophils
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Splenocytes or isolated bone marrow neutrophils were seeded into 96-well plates (1–2 × 106 per well). Cells were centrifuged for 5 min at 350 RCF; then, they were stained with Fixable Viability Stain 440UV (565388; BD Horizon) according to the manufacture’s recommendation and incubated for 6 min at 37 °C, 5% CO2. After two washes, the samples were blocked with 5% FBS in PBS for 10 min at RT. Subsequently, surface antigens were labelled using the following antibodies: anti-CD11b BV510 (562950; BD Horizon; 1:400), anti-CD45 V500 (562129; BD Horizon; 1:200), anti-Ly6G BV605 (563005; BD Horizon; 1:200), anti-Ly6C BV711 (128037; BioLegend; 1:400), anti-CD80 Pe-Cy7 (104734; BioLegend; 1:200), anti-CD83 APC (558206; BD Pharmingen; 1:200), anti-I-A/I-E BB700 (746197; BD OptiBuild; 1:200) and anti-CD86 FITC (561962; BD Pharmingen; 1:200) for 30 min at RT. Cells were then fixed using BD Cell fix and analysed using a BD LSRFortessa flow cytometer.
5
Cytokine and Antibody Stimulation of Immune Cells
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Recombinant human (rh) cytokines, including IL-1α, IL-2, IL-4, GM-CSF, IFN-γ, and TNF-α, and anti-CD3 and anti-CD28 antibodies were purchased from Beijing T&L Biotechnology. Fluorophore-labeled primary antibodies, including anti-CD3-FITC, anti-CD4-phycoerythrin/PE, anti-CD56-allophycocyanin/APC, anti-CD80-PE, anti-CD83-APC, and anti-CD86-PerCP-Cy5.5, were acquired from BD Biosciences; anti-CD44 and anti-EpCAM antibodies were obtained from Abcam. Ficoll-Paque PLUS medium was obtained from GE Healthcare Life Sciences; Lonza X-VIVO™ 15 medium from Fisher Scientific; CCK-8 reagent from Dojindo Molecular Technologies; and TRIzol reagent from Molecular Research Center.
6
Flow Cytometry Analysis of Cell Markers
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Flow cytometry was performed as previously described [26 (link),27 (link)]. Analysis of the expression levels of cell-surface markers was performed by flow cytometry. DCs were blocked for 5 min with PBS containing 10% heat inactivated human AB serum (Sigma-Aldrich, St. Louis, MO, USA), and then stained by adding the antibodies to the same buffer. All other cell types were stained on PBS containing 3% FBS. Cells were labeled with anti-CD1a-PE, anti-CD3-PC7, anti-CD4-FITC, anti-CD14-PC7, anti-CD19-ECD, anti-CD25-PC7, anti-CD69-PC5, anti-CD80-FITC, anti-CD86-PC5.5, anti-CD86-PE, anti-HLA-DR-ECD, anti-HLA-DR-PB, anti-HLA-DR-PE (all from Beckman Coulter Inc., Brea, CA, USA), anti-CD8-Pacific Blue, anti-CD71-APC-Cy7, anti-CD197-APC-Cy7 (CCR7) (all from BioLegend, San Diego, CA, USA), anti-CD83-APC (BD Biosciences, Franklin Lake, NJ, USA), and anti-CD3-PO (Abcore, Ramona, CA, USA).
Levels of surface expression on cells were estimated by flow cytometry (Gallios; Beckman Coulter, Brea, CA, USA) and analyzed using Kaluza software (Beckman Coulter, Brea, CA, USA). Entry of FITC-labeled p24 peptide into the DCs was also estimated by flow cytometry. DCs were collected 2 h, 24 h, and 48 h after complex addition, washed with PBS, and acid washed, in order to eliminate any peptide attached to the cell surface. Fluorescence was measured by flow cytometry.
Levels of surface expression on cells were estimated by flow cytometry (Gallios; Beckman Coulter, Brea, CA, USA) and analyzed using Kaluza software (Beckman Coulter, Brea, CA, USA). Entry of FITC-labeled p24 peptide into the DCs was also estimated by flow cytometry. DCs were collected 2 h, 24 h, and 48 h after complex addition, washed with PBS, and acid washed, in order to eliminate any peptide attached to the cell surface. Fluorescence was measured by flow cytometry.
7
Phenotypic Analysis of Dendritic Cells and Macrophages
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Analysis of cell-surface phenotype of DCs and MØs was performed by flow cytometry. Cells were labeled with anti-CD4-FITC, anti-CD69-PC5, anti-CD80-FITC, anti-CD1a-PE, anti-HLA-DR-ECD, anti-CD14-PC7, anti-CD86-PC5.5 (all from Beckman Coulter Inc., Brea, CA, USA), anti-CD8-Pacific Blue, anti-CD36-APC, anti-CD197-APC-Cy7 (CCR7) (all from BioLegend, San Diego, CA, USA), anti-CCR5-PE, anti-CD209-PE, anti-CD83-APC (all from BD Biosciences, Franklin Lake, NJ, USA), anti-CXCR4-APC (RandD systems, Minneapolis, MN, USA) and anti-CD163-FITC (MBL International Corp., Woburn, WA, USA). Levels of surface expression on cells were estimated by flow cytometry (Gallios; Beckman) and analyzed using Kaluza software (Beckman).
8
Monoclonal Antibody Characterization of MoDC and γδ T Cells
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The following monoclonal antibodies were used to characterize MoDC: anti-CD86 FITC, anti-CD1a PE, anti-HLA-DR PERCP, anti-CD83 APC, anti-CD14 APC-H7, anti-CD80 FITC, anti-CD40 PE, anti-HLA-I APC, anti-CCR7 PE-Cy7, anti-CCR5 APC-H7 from BD Biosciences, and anti-BT3A.1 PE from BioLegend. To evaluate γδ T lymphocytes phenotype we used anti-Vδ2 FITC, anti-CD3 PE, anti-CD69 PERCP, anti-CD45RA PE-Cy7, anti-CD27 APC, anti-CD16 PACIFIC BLU, anti-CD25 APC-H7 (BD Biosciences). In brief, the cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide, and were stained with the mAbs for 15 min at 4°C. The cells were then washed and fixed with 4% paraformaldehyde, and analyzed using a FACS Canto II (Becton Dickinson). Since the presence of 2 purified populations, the gating strategy was performed as follow: dead cells were excluded by scatter characteristics; MoDC were identified by morphological parameters (FSC vs SSC); gated cells were then analyzed for the expression of the molecules described above. T lymphocytes were first gated by using morphological parameters; in this gate Vγ9Vδ2 T cells were identified as Vδ2+CD3+. Analysis was carried out by using Facs Diva software (Becton Dickinson). The histogram overlays were performed by FlowJo software (TreStar, Olten, Switxìzerland).
9
Multicolor Flow Cytometry Analysis
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CRC lines, cells separated from human CRC tissue fragments, DCs and effector leukocytes (mentioned in previous paragraphs) were stained with the following cocktail of monoclonal antibodies purchased from BD Biosciences, USA: anti-CD29-APC (clone MAR4, IgG1κ), anti-CD44-FITC (clone C26, IgG2bκ), anti-CD95-PE (clone DX2, C3H/Bi IgG1κ), anti-FasL Biotin (clone NOK-1, IgG1) coupled with Streptavidin-APC, anti-CD3-PE (clone UCHT1, IgG1κ), anti-CD4-FITC (clone PA-T4, IgG1κ), anti-CD11c-APC (clone S-HCL-3, IgG2b),anti-CD14-PerCP (clone MøP9, IgG2b), anti-CD25-PE (clone M-A251, IgG1κ), anti-CD56 (clone B159, IgG1κ), anti-CD80-PE (clone L307, IgG1κ), anti-CD83-APC (clone HB15e, IgG1κ), anti-HLA-DR-PerCP (clone L243, IgG2a). Anti-CD133/2-PE (clone 293C3, IgG2bκ) monoclonal antibodies were purchased from MiltenyiBiotec. After 30 min incubation in the dark, samples were fixed with 1% PFA or PBS + 1 mM EDTA for adherent or spherical cells, respectively, and prepared for further analysis. Flow cytometric analysis was performed using FACS Calibur flow cytometer (BD Biosciences, USA) with BD CellQuest Pro Software.
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