For analysis, the cells were harvested and incubated with fluorescently labeled anti-human antibodies for 30 min at 4 °C in the dark. Each sample was stained by 1 ul of each antibody at a concentration of 0.2 mg/mL in 100 ul of PBS containing 2% FBS. In flow cytometry, live aAPCs or CD3+ Jurkat cells were gated and recorded in at least 1 × 104 cells to determine the GFP expression rate. The following antibodies were used to detect targeting molecules: anti-CD80-PE (BioLegend, San Diego, CA, USA), anti-CD83-PE (BioLegend, San Diego, CA, USA), anti-CD137L-PE (BioLegend, San Diego, CA, USA), anti-CD-32-FITC (BioLegend, San Diego, CA, USA), anti-CD9-PE (BioLegend, San Diego, CA, USA), anti-CD63-PE (BioLegend, San Diego, CA, USA), anti-CD81-PE (BioLegend, San Diego, CA, USA), anti-CD8α-PerCP-cy5.5 (BioLegend, San Diego, CA, USA), and anti-CD3-BV421 (BioLegend, San Diego, CA, USA). Fluorescence was measured using a BD FACS Canto (BD Biosciences) and analyzed using the FlowJo v10 software (BD Biosciences).
Anti cd83 pe
Manufactured by BioLegend
Sourced in United States
Anti-CD83-PE is a fluorescent-labeled antibody that binds to the CD83 protein. CD83 is a cell surface marker expressed on mature dendritic cells and some activated T cells. This product can be used for the detection and analysis of CD83-positive cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11c apc, by BioLegend (3 mentions)
Facscalibur, by BD (2 mentions)
Recombinant human il 4, by R&D Systems (2 mentions)
Anti cd1a fitc, by BioLegend (2 mentions)
Gm csf, by R&D Systems (2 mentions)
Facscanto, by BD (1 mentions)
Anti cd80 pe, by BioLegend (1 mentions)
Anti cd3 bv421, by BioLegend (1 mentions)
Anti cd63 pe, by BioLegend (1 mentions)
Anti cd81 pe, by BioLegend (1 mentions)
Anti cd8α percp cy5, by BioLegend (1 mentions)
Anti cd 32 fitc, by BioLegend (1 mentions)
Anti cd9 pe, by BioLegend (1 mentions)
Anti cd40 pe, by BioLegend (1 mentions)
Anti pd l1 apc, by BioLegend (1 mentions)
Human trustain fcx solution, by BioLegend (1 mentions)
Anti cd86 pacific blue, by BioLegend (1 mentions)
Anti cd14 af700, by BioLegend (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Anti cd25 pe, by BD (1 mentions)
9 protocols using anti cd83 pe
1
Phenotypic Characterization of aAPCs
2
Phenotyping of Monocyte-Derived Dendritic Cells
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Phenotyping of moDC was performed by flow cytometry using anti-CD83-PE and anti-CD1a-FITC specific antibodes from BioLegend at the protein level (San Diego, CA, USA). Fluorescence intensities were measured with FACSCalibur (BD Biosciences). Data analysis was performed with the FlowJo software (Tree Star, Ashland, OR, USA).
3
Characterizing Dendritic Cell Phenotype
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Before staining, FcγR were blocked using the Human TruStain FcX Solution (BioLegend) for 10 min at room temperature in PBS 2% FBS. Cells were then stained for 15 min at 4°C in PBS 2% FBS with one or more of the following Ab: anti- CD14-AF700 (BioLegend), anti-DC-SIGN-AF647 (BioLegend), anti-CD1a-AF488 (BioLegend), anti-CD83-PE (BioLegend), anti-CD86-Pacific Blue (BioLegend), anti-CD40 PE (BioLegend), anti-CD80 Pacific Blue (BD Biosciences), anti-PD-L1 APC (BioLegend), anti HLA-DR A700 (BioLegend).
4
Multiparametric Flow Cytometry Analysis
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All cells were labeled with live/dead dye near infra red (Life Technologies) for dead cell exclusion and treated with Fc Block (Miltenyi, San Diego, CA, USA) prior to staining with fluorescently labeled antibodies. Anti-human antibodies used for DC staining were anti-CD83 PE (Biolegend, San Diego, CA, USA), anti-CD86-PE-Cy7 (Biolegend), anti-HLA-DR V450 (BD), and anti-CD11c APC (BD). Antibodies used in the T cell characterization were anti-LAG-3 FITC (Novus, Littleton, CO, USA), anti-PD-1 PerCP-Cy5.5, anti-CD3 Alexa700, anti-CD4 Brilliant Violet 650, anti-CD8 Brilliant Violet 570, anti-IFN-γ PE, anti-IL-5, anti-CD62L PE, and anti-CD45RA Alexa700 (all, Biolegend), anti-CD25 PE, and anti-IFN-γ PE-Cy7, anti-IL-4 PE-Cy7 (all BD), and anti-TIM-3 APC (R&D Systems). Surface staining and intracellular staining was performed using Cytofix/Cytoperm™ Plus kit (BD) according to the manufacturer’s instructions. Data acquisition was performed using the LSRFortessa flow cytometer (BD). Data analysis was performed with FlowJo version 9 (Tree Star, Inc., Ashland, OR, USA).
5
Phenotypic Analysis of Dendritic Cells
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DC phenotype was analyzed by flow cytometry. Briefly, DCs were harvested, washed in phosphate-buffered saline (PBS) and resuspended at 106 cells/ml. Each experimental sample was of 2 × 105 cells/tube (BD Biosciences, NJ, USA), cells were incubated with specific fluorochrome-conjugated antibodies for 30 min at 4°C in the dark. After washing in PBS (two times), the cells were analyzed. The following monoclonal antibodies (mAb) were used: anti-HLAII-DR-APCH7 and anti-CD86-Pecy7, from BD Biosciences; anti-CD14-BB700, anti-CCR7-AlexaFlour647, anti-CD83-Pe, anti-CD40-BB515, anti-ICOS-L-Pe from BioLegend (San Diego, CA); and anti-VEGFR-1-PE from R&D Systems. MoAbs anti-IgG1-BB515, -PE, -Pecy7, and -BB700; -AlexaFluor647; and -APC-H7 (BioLegend) were used as isotype controls. All mAbs were purchased from BD Biosciences and BioLegend. Flow cytometry analysis was performed using FACSCanto II flow cytometer running FACS Diva data acquisition and analysis software (BD Biosciences).
6
Generation of Dendritic Cells from Monocytes
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DCs were generated from monocytes isolated from PBMC. Purified monocytes (1 ×
106 cells/mL) were resuspended in RPMI 1640 containing human
recombinant IL-4 (80 ng/mL) and GM-CSF (80 ng/mL) (R&D Systems, USA) for 7
days at 37°C and 5% CO2 [29 (link),
30 (link)]. Then, cells were incubated with
mAbs (Biolegend, USA) anti-CD14-PerCP-Cy 5.5 (0.3 μL), anti-CD1a-FITC (1 μL),
anti-CD83-PE (1 μL) and anti-CD11c-APC (1 μL) for 30 min. A Fluorescence Minus
One (FMO) control was included.
This phenotyping protocol was performed to assure the cell differentiation and
analyzed in a flow cytometer model FACS CaliburTM (BD Becton Dickinson and
Company, USA). A total of 50.000 events were acquired and the expression of
following cell surface markers was analyzed:
CD14low/CD1ahigh/CD11chigh/CD83low[31 (link)].
DCs were incubated with propolis alone or in combination with MAGE-1 and RA for
48 h.
106 cells/mL) were resuspended in RPMI 1640 containing human
recombinant IL-4 (80 ng/mL) and GM-CSF (80 ng/mL) (R&D Systems, USA) for 7
days at 37°C and 5% CO2 [29 (link),
30 (link)]. Then, cells were incubated with
mAbs (Biolegend, USA) anti-CD14-PerCP-Cy 5.5 (0.3 μL), anti-CD1a-FITC (1 μL),
anti-CD83-PE (1 μL) and anti-CD11c-APC (1 μL) for 30 min. A Fluorescence Minus
One (FMO) control was included.
This phenotyping protocol was performed to assure the cell differentiation and
analyzed in a flow cytometer model FACS CaliburTM (BD Becton Dickinson and
Company, USA). A total of 50.000 events were acquired and the expression of
following cell surface markers was analyzed:
CD14low/CD1ahigh/CD11chigh/CD83low[31 (link)].
DCs were incubated with propolis alone or in combination with MAGE-1 and RA for
48 h.
7
Generating Dendritic Cells from Monocytes
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DCs were generated from human monocytes after PBMC isolation. Purified monocytes (1×106 cells/mL) were resuspended in complete RPMI 1640 medium plus human recombinant IL-4 (80 ng/mL) and GM-CSF (80 ng/mL) (R&D Systems, USA) for seven days at 37°C and 5% CO2 (19 (link),20 (link)). Cells were then incubated with anti-CD14 mAbs conjugated with PerCP/Cy5.5 (0.3 μL), anti-CD1a-FITC (1 μL), anti-CD83-PE (1 μL), and anti-CD11c-APC (1 μL) for 30 min (Biolegend). A Fluorescence Minus One (FMO) control was performed. DCs phenotyping protocol was carried out to confirm cell differentiation, and cells were analyzed in the FACS CaliburTM flow cytometer acquiring a total of 50,000 events. DCs were accordingly generated, presenting the typical cell markers CD11chigh, CD1ahigh, CD83low, and CD14low (21 (link)).
DCs were incubated with propolis alone or in combination with EtxB or LPS diluted in RPMI 1640 supplemented with 10% FBS (complete medium) for 48 h in the following protocols.
DCs were incubated with propolis alone or in combination with EtxB or LPS diluted in RPMI 1640 supplemented with 10% FBS (complete medium) for 48 h in the following protocols.
8
Multicolor Flow Cytometry for Immune Profiling
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The antibodies used were anti-HLA-DR-FITC, mouse-IgG2b-FITC (both from BD Biosciences, San Jose, CA, USA); anti-CD83-PE, mouse-IgG1-PE (both from BioLegend, San Diego, CA, USA); anti-CD14-PerCP, mouse-IgG1k-PerCP, anti-CD11-APC, mouse-IgG1k-APC, biotinylated antihuman DEC-205 monoclonal antibody, biotinylated mouse IgG2bk (all from eBiosciences, San Diego, CA, USA); antihuman interferon (IFN)-γ enzyme-linked immunosorbent assay kit (BD Pharmingen, San Diego, CA, USA).
9
Phenotypic Analysis of CD11c+ DCs
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CD11c+DCs were incubated at 4°C for 30 minutes with the following fluorochrome-labeled anti-mouse monoclonal antibodies: anti-CD11c-APC (Biolegend, USA), anti-CD74-FITC (BD Biosciences, USA), anti-CD83-PE (Biolegend, USA) and anti-CD86-PE (Biolegend, USA). Samples were acquired on a FACS Calibur flow cytometer (BD Biosciences, USA) and data were analyzed with Flowjo software (BD Biosciences, USA).
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