Upon treatment, BDCA-1+ myDCs were seeded in a 96-well round bottom plate at a density of 150,000–200,000 cells per well. BDCA-1+ myDCs were treated with either active T-VEC (MOI 10), heat-inactivated T-VEC (MOI 10; heat-inactivation: 15 min 65 °C, 1 min 100 °C) or conditioned medium, i.e., SN of dying 624-mel and 938-mel 24 h and 48 h after treatment with T-VEC (MOI 1). Untreated BDCA-1+ myDCs served as a negative control. As a positive control, BDCA-1+ myDCs were treated with a mix containing ssRNA fragments derived from HIV-1 long terminal repeat and protamine sulfate (PS/LTR). After overnight incubation, cells were harvested and stained with anti-CD11c-AlexaFluor 700 (BD Biosciences, 561352), anti-CD1c-BV510 (BD Biosciences, 742747), anti-CD80-PerCP-eFluor710 (ThermoFisher Scientific, 46-0809-42), anti-CD86-BV421 (BD Biosciences, 562432), anti-CD40-APC (BioLegend, 334310), anti-CD83-PE (BD Biosciences, 556855), anti-CD274-PE-CF594 (BD Biosciences, 563742) and anti-HLA-ABC-FITC (BD Biosciences, 557348) for 20 min at 4 °C and in the dark. Cells were acquired on the flow cytometer (BD LRS Fortessa) and data were analyzed with with FlowLogic software (Miltenyi Biotec, Version 7.3).
Anti cd83 pe
Manufactured by BD
Sourced in Spain, United States
Anti-CD83-PE is a monoclonal antibody conjugated with the fluorescent dye phycoerythrin (PE). CD83 is a cell surface glycoprotein that is expressed on mature dendritic cells and can be used as a marker for this cell type. The Anti-CD83-PE product can be used to identify and analyze CD83-positive cells in various applications such as flow cytometry.
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Lab products found in correlation
Anti cd80 pe, by BD (5 mentions)
Anti cd86 fitc, by BD (4 mentions)
Anti cd86 pe, by BD (4 mentions)
Anti hla abc pe, by BD (3 mentions)
Anti cd45 fitc, by BD (2 mentions)
Anti hla dr pe, by BD (2 mentions)
Anti cd40 fitc, by BD (2 mentions)
Anti cd40 pe, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Flowlogic software, by Miltenyi Biotec (1 mentions)
Anti cd86 bv421, by BD (1 mentions)
Anti cd40 apc, by BioLegend (1 mentions)
Anti hla abc fitc, by BD (1 mentions)
Anti cd11c alexafluor700, by BD (1 mentions)
Anti cd8 v500, by BD (1 mentions)
Anti cd3 pacific blue, by BD (1 mentions)
Anti cd11c pe, by BD (1 mentions)
Anti cd14 fitc, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
11 protocols using anti cd83 pe
1
Activated myDCs response to T-VEC
2
CD8 T Cell-Dendritic Cell Interaction
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Epitope-specific CD8 T cells were added directly to autologous iDC at 3:1 effector to target (E/T) ratio in the presence or absence of peptide of interest (10 μM) and co-cultured for 48 hours. Immature dendritic cells cultured in the presence of a maturation cocktail containing TNFα (50ng/ml), IFNα (3000U/ml), IFNγ (1000U/ml), IL-1B (25ng/ml), and pI:C (20ug/ml) were used as a positive control. After two days, cells were stained with dead cell dye (Invitrogen), anti-CD3-Pacific Blue, anti-CD8-V500, anti-CD14-alexa700, anti-CD83-PE, and anti-CD86-FITC (all from BD Pharmingen) at 4°C for 30 min. Cells were then washed and events were acquired on an LSR II flow cytometer.
3
Flow Cytometry Analysis of Dendritic Cells
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A subset of six mice per group was used for flow cytometry analysis. Cell suspensions were incubated with a mixture of antibodies (anti‐CD11c‐PE, anti‐CD45‐FITC, anti‐CD83‐PE, anti‐CD86‐FITC; BD Biosciences) at 4°C for flow cytometry analysis to determine the percentage of DCs in hearts, spleens and peripheral blood, respectively. Samples were loaded after two washes with PBS (2% BSA), and the raw data were analysed performed with a flow cytometer (Beckman).
4
Characterization of moDC and mDC Responses
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6×105 mDCs or moDCs were cultured for 18 h at 37°C in 200 µL CellGro GMP DC medium (CellGenix, Cat. No.: 20801-0500) supplemented with 50 µg/mL AfuLy, 1-5 µg/mL Aspergillus proteins CcpA or Shm2, 1 µg/mL LPS (Sigma, positive control), or plain medium (negative control). Thereafter, DCs were stained with anti-CD1a-APC (moDCs) or anti-CD1c-APC (mDCs), anti-CD14-FITC, anti-CD80-APC, anti-CD83-PE, anti-CD86-FITC, anti-CD40-FITC, anti-HLA-ABC-PE, anti-HLA-DR-PE (BD), and anti-CCR7-APC (Miltenyi Biotec) in three different panels. Samples were analyzed using a FACS Calibur cytometer (BD), Cell Quest Pro software (BD), and Flow Jo software (version 10.6.2.). Dead cells were excluded from analysis by light scatter (FSC/SSC) properties.
5
Phenotypic Analysis of Electroporated Murine Dendritic Cells
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mDCs were electroporated as described above, incubated in DC medium at 37°C in a humidified incubator, and harvested 24 h after electroporation. The expression of distinct markers was analyzed by flow cytometry. For the determination of surface marker expression, the following antibodies and their respective isotype controls were used: IgG1-PE, anti-CD25-PE, anti-CD40-PE, anti-CD70-PE, anti-CD80-PE, anti-CD83-PE, anti-CD86-PE (all from BD), and IgG3-PE (eBioscience). Seventy-five to one hundred thousand cells were incubated with antibody for 30 minutes at 4°C in FACS solution, consisting of PBS supplemented with 1% FCS (PAA, GE healthcare) and 0.02% sodium azide (Merck). The cells were then washed once with FACS solution and immunofluorescence was measured using a FACScan cytofluorometer equipped with CellQuest software (BD Biosciences). mDCs were gated on in the forward and side scatter channels and the mean fluorescence intensities (MFIs) were measured. Specific MFI was calculated by subtraction of the MFI of the isotype control.
6
Phenotypic Characterization of Dendritic Cells
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DC phenotype staining was performed using the following antibodies directly conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE): IgG1-FITC and IgG1-PE as isotype controls (both from Biolegend); anti-HLAII-DR-FITC, anti-CD86-FITC, anti-CD83-PE (all from BD Biosciences), anti-CD40-PE, anti-CD14-PE, and anti-CCR7-FITC (all from Biolegend). DCs (2 × 105 cells/50μL sample) were incubated with conjugated MoAb (according to the manufacturer's recommendation) for 30 min at room temperature (RT). After washing (in 2 mL of PBS w/o Mg++ and Ca++, centrifuged at 250 × g for 5 min), cell pellet was resuspended in PBS (100 μL); at least 1 × 104 events were evaluated using a FACSCanto II flow cytometer running FACSDiva data acquisition and analysis software (Becton Dickinson).
To evaluate MUC1 expressed by MUC1-DG75 cells, 1 × 105 cells were incubated with MoAb Ma552 (1:40; Monosan, Netherlands, 50 μL/sample) for 30 min at RT and binding revealed with FITC-conjugated anti-mouse antibody (1:600; Jackson-Immunoresearch Laboratories, PA, USA). MoAb MOPC21 (1:100; Sigma-Aldrich, 50 μL/sample) was employed as isotype control.
To evaluate MUC1 expressed by MUC1-DG75 cells, 1 × 105 cells were incubated with MoAb Ma552 (1:40; Monosan, Netherlands, 50 μL/sample) for 30 min at RT and binding revealed with FITC-conjugated anti-mouse antibody (1:600; Jackson-Immunoresearch Laboratories, PA, USA). MoAb MOPC21 (1:100; Sigma-Aldrich, 50 μL/sample) was employed as isotype control.
7
Immunophenotyping of Monocyte Subsets
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The expression level of relevant markers of CD14+CD16− monocytes and their descendants’ under various culturing systems was determined by flow cytometry using specific antibodies. Briefly, cells were incubated with saturating concentration of human serum for 10 min at 4°C to prevent unspecific binding to Fcγ receptors and stained with mouse anti-CD45 FITC, anti-CD14 APC, anti-CD1a PE, anti-CD16 PE, anti-CD32 PE, anti-CD40 PE, anti-CD80 PE, anti-CD86 PE, anti-CD83 PE, anti-CD163 PE, anti-CD206 PE, anti-Toll-like receptor type 2 (TLR2) PE (BD Biosciences, Madrid, Spain), anti-HLA class II PE (eBioscience), and anti-CD64 PE (Life Technologies) monoclonal antibodies (Table S1 in Supplementary Material) for 30 min at 4°C. Cells were washed and then stained with 7-aminoactinomycin D (7-AAD) to exclude dead cells. Matched isotypes were used as negative controls. Cells were acquired using BD Fortessa flow cytometer (BD Biosciences) and analyzed using FACSDiva 8 (BD Biosciences) and FCS Express 6 (De Novo software, CA, USA) software. The expression of various markers is presented as the percentage of positive cells (%) and geometric mean fluorescence (Geometric mean).
8
Quantifying Human Dendritic Cell Markers
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Anti-HLA-DR-FITC, anti-CD40-FITC, anti-CD83-PE, anti-CD80-FITC, and anti-CD86-PE (BD Biosciences Pharmingen) were used under standard surface marker labeling procedure to label their corresponding functional surface molecules on DC surface. All of the assigned DC groups were labeled with the aforementioned antibodies and read using flow cytometry (Beckman Coulter Epics Altra). anti-CD40-FITC was co-labeled together with anti-CD83-PE, while anti-CD40-FITC was co-labeled with CD83-PE. PE fluorescence spillover compensation was done on FITC channel at 6.5%. FITC fluorescence spillover compensation was done on PE channel at 12.5%. This observation method was applied on three technical replicates from a single donor on different flow cytometry runs.
9
Phenotypic Characterization of DCs and PBMCs
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The DCs and PBMCs that were obtained were collected and suspended (1-5 x 10 6 /mL) using anti-CD14 PE, anti-CD83 PE, anti-CD80 PE, and anti-CD86 PE (BD Bioscience). Cell phenotype was determined by flow cytometry (Agilent Biosciences) and the results were digitized with the ACEA NovoExpress program.
10
Phenotypic Analysis of PLGA Particle Uptake
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Uptake of PLGA particles and the phenotype of mDCs and pDCs were determined by flow cytometry. Plasmacytoid DCs and mDCs were cultured overnight with different concentrations of PLGA particles containing PFC and atto-647. The following primary monoclonal antibodies (mAbs) and appropriate isotype controls were used: anti-BDCA2-PE, anti-CD123-APC, anti-CD11c-PE and anti-BDCA-1-APC (all Miltenyi Biotec); anti-HLA-ABC-PE, anti-HLA-DR/DP-FITC, anti-CD80-PE, anti-CD83PE, anti-CD86-APC (all BD Bioscience Pharmingen, CA, USA); anti-CD40-PE (Beckman Coulter, Mijdrecht, The Netherlands). Cells were analyzed by flow cytometry on a FACSCalibur (BD Biosciences, CA, USA).
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