Dendritic cells were stimulated with inactivated A. fumigatus conidia and germ tubes (MOI = 1), 100 µg/ml zymosan depleted [a yeast cell wall preparation, which was treated with hot alkali to remove all TLR-stimulating properties to selectively activate Dectin-1 (dZym, InvivoGen)] or 1 mg/ml lipopolysaccharid (LPS, Sigma) for 24 h. Subsequently, cells were harvested, washed, and resuspended in cold Hank’s balanced salt solution (HBSS, Sigma) containing 2 mM EDTA (Sigma). The following antibodies were used for extracellular staining: HLA-ABC-PE (BD Biosciences), HLA-DR-PE (BD Biosciences), CD1a-APC (BD Biosciences), CD14-FITC (BD Biosciences), CD80-APC (Miltenyi Biotec), CD86-PE (BD Biosciences), Dectin-1-PE (R&D) (human cells) and HLA-2Kb-FITC (BD Biosciences), HLA-Ia/I-E-PE (BioLegend), CD11c-APC (BioLegend), CD80-FITC (BioLegend), CD86-PE (BD Biosciences), and Dectin-1-PE (R&D) [murine cells]. 5 × 105 cells were stained for 15 min at 4°C. Subsequently, cells were washed twice and 104 viable cells according to FSC-SSC properties were acquired using a FACS Calibur (BD Biosciences) flow cytometer and CellQuest Pro software (version 5.2). Data were analyzed with FlowJo software (Tree Star Inc., Ashland, OR, USA).
Cd86 pe
Manufactured by BD
Sourced in United States, Germany, United Kingdom
CD86-PE is a fluorescently labeled antibody that binds to the CD86 cell surface marker. CD86 is a costimulatory molecule expressed on antigen-presenting cells and plays a role in T cell activation. The PE fluorescent label allows for the detection and quantification of CD86-positive cells using flow cytometry.
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Lab products found in correlation
Cd80 pe, by BD (15 mentions)
Cd80 fitc, by BD (11 mentions)
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Cd83 apc, by BD (6 mentions)
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Cd206 apc, by BD (6 mentions)
Hla dr fitc, by BD (6 mentions)
Cd11c apc, by BD (6 mentions)
Cd4 pe, by BD (6 mentions)
Cd14 fitc, by BD (5 mentions)
Facscanto 2, by BD (5 mentions)
Cd69 fitc, by BD (5 mentions)
Facsdiva software, by BD (5 mentions)
Hla dr pe, by BD (4 mentions)
Cd4 fitc, by BD (4 mentions)
Cd40 pe, by BD (4 mentions)
Cd3 fitc, by BD (4 mentions)
Cd11b fitc, by BD (4 mentions)
Cd11c fitc, by BD (4 mentions)
Facscalibur flow cytometer, by BD (4 mentions)
81 protocols using cd86 pe
1
Dendritic Cell Immunophenotyping Assay
2
Immunophenotyping of Murine and Human Dendritic Cells
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For direct immunofluorescence staining of murine DC, the following phycoerythrin (PE)-conjugated rat anti-mouse antibodies were used (BD Biosciences Pharmingen): CD11c-PE (dilution 1:100), CD80-PE (dilution 1:600), CD86-PE (dilution 1:600), and CD40-PE (dilution 1:200). For direct cell staining, 50 μl of cell suspension was mixed with 50 μl of diluted antibody, and the mixture was incubated at 4 °C for 45 min. For indirect cell staining, 50 μl of cell suspension was incubated with an equal volume of unconjugated mouse anti-mouse I-A and MHC II (dilutions for both, 1:100) antibodies for 30 min at 4 °C, washed, and further incubated at 4 °C for 45 min with AlexaFluor 488-conjugated rat anti-mouse antibody (dilution 1:400; BD Biosciences Pharmingen).
Human dendritic cells were labeled directly with monoclonal antibodies against CD11c-APC (dilution 1:100), HLA-DR-FITC (dilution 1:50), CD83-APC (dilution 1:50), CD86-PE (dilution 1:100), CD1a-FITC (dilution 1:50) (BD Biosciences Pharmingen). For cell staining, 50 μl of cell suspension was mixed with 50 μl of diluted antibody, and the mixture was incubated at 4 °C for 45 min. Appropriate isotype antibodies were used as controls to determine non-specific binding. Cells were analyzed using a FACSCalibur flow cytometer (Becton-Dickinson, USA).
Human dendritic cells were labeled directly with monoclonal antibodies against CD11c-APC (dilution 1:100), HLA-DR-FITC (dilution 1:50), CD83-APC (dilution 1:50), CD86-PE (dilution 1:100), CD1a-FITC (dilution 1:50) (BD Biosciences Pharmingen). For cell staining, 50 μl of cell suspension was mixed with 50 μl of diluted antibody, and the mixture was incubated at 4 °C for 45 min. Appropriate isotype antibodies were used as controls to determine non-specific binding. Cells were analyzed using a FACSCalibur flow cytometer (Becton-Dickinson, USA).
3
Multicolor Flow Cytometry Immunophenotyping
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Non-specific binding sites on harvested cells were blocked with 20% inactivated AB-serum in PBS containing 0.1% BSA and 0.1% NaN3 for 20 min at 4 °C. Then, cells were stained for 30 min at 4 °C with fluorescence-labelled antibodies: CD83-FITC, CD19-PECy7, CD80-APC647, CD14-APCCy7, HLA-DR-Brilliant Violet 421 (all Biolegend, San Diego, CA) CD86-PE, CD86-PECy7 (both BD Biosciences) and fixable viability dye eFluor506 (eBioscience, San Diego, CA, USA). Fluorescence was assessed by flow cytometry on a BD FACS Canto II and analyzed with FlowJo software.
4
Multiparameter Phenotyping of Dendritic Cells and T Cells
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Directly conjugated mAbs against CD1c-BV421 (BD Biosciences), HLA-DR-PERCP (Invitrogen, Waltham, MA, USA), B7H3(CD276)-APC (Miltenyi, Stockholm, Sweden), B7H1-PE (Invitrogen), NOS1-FITC (Santa Cruz Biotechnology, Dallas, TX, USA), PDL2-APC (R&Dsystems), HVEM-PE (Invitrogen), Arginase 1-PerCP-eFluor 710 (Thermo fisher, Waltham, MA, USA), IDO-FITC (R&Dsystems), CD80-APC (Miltenyi), CD86-PE (BD Biosciences), CD85d (ILT4)-PerCP-eFluor 710 (Thermo fisher), COX2-FITC (Invitrogen), CD30L/TNFSF8-APC (R&Dsystems), Galectin-9-PE (Miltenyi), and B7-H4-Alexa Fluor 700 (R&Dsystems) were used for DC and T cell phenotyping before and after the DC–T cell interaction in the coculture. In brief the cells in the coculture were harvested, washed, and stained with the mAbs for 20-30 min at 4°C. Thereafter the cells were fixed with 4% PFA (Sigma Aldrich), permeabilized with 0.2% Saponin (Sigma Aldrich), and intracellular staining was performed at 4°C for 30-45 min. Data was acquired using FACS canto II, and the FlowJo software v9 (Treestar, OR, USA) was used for data analysis.
5
Oxymatrine Modulates Dendritic Cell Activation
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Cells were treated with lipopolysaccharide (LPS; 100 ng/ml), OMT (1 mg/ml) and OMT (1 mg/ml) + LPS (100 ng/ml) for 48 h. According to preliminary experiments in the present study, oxymatrine inhibited the proliferation of dendritic cells, and the effect of inhibiting proliferation was observed at OMT concentration of 0.8 mg/ml. However, the inhibitory effect was not significantly increased with the increase of concentration, so the concentration of OMT in this experiment was chosen to be 1.0 mg/ml (data not shown). The cultured DCs were collected and centrifuged at 1,000 × g for 5 min at 4°C. The supernatant was discarded and the cells were washed three times with PBS. In total, 5 µl CD83 antigen (CD83)-phycoerythrin (PE; 556855; BD Biosciences, San Jose, CA, USA), T-lymphocyte activation antigen CD86 (CD86)-PE (560957; BD Biosciences), CD11 antigen-like family member C (CD11c)-PE (555392; BD Biosciences) and major histocompatibility complex II (MHC II)-PE(555812, BD Biosciences) were added to each tube. Subsequent to gentle mixing, the cells were incubated at room temperature in the dark for 30 min. The cells were washed twice with PBS and resuspended in 100 µl PBS, and the DC surface markers were detected using a flow cytometer (BD Accuri C6; BD Biosciences).
6
Characterizing DC Surface Phenotypes
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To characterize the cell surface phenotypes on DCs, flow cytometry was performed using a FACSAria cell sorter (Becton Dickinson, San Jose, CA, USA) after labeling of the cells with CD86-PE, CD83-FITC, CCR7-FITC (PharMingen, San Diego, CA, USA), and the relevant isotype controls (mouse IgG1 and IgG2a, PharMingen). Cell debris was eliminated from the analysis by forward and side-scatter gating, and the data were analyzed with WinMDI Version 2.9 software (Biology Software Net).
7
Phenotypic Analysis of T Cells
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For phenotypic analysis of T cells, the PPD and peptides stimulated lungs and spleen/LNs cells were analysed by flow cytometry. Lymphocytes culture were harvested and stained with fluorochrome tagged anti-CD4-PE, CD8-APCCy7, CD62L-FITC, CD44-PerCPCy5.5, CD11c-PECy7, F4/80-APC, CD86-PE, CD80-FITC, CD40-PECy5, and MHC-II-PerCPCy5.5abs (BD Biosciences, San Jose, CA). Briefly, lymphocytes were harvested in tubes and washed with FACS buffer (PBS + 2%FCS). Cells were Fc blocked using anti-mouse CD16/CD32 Ab. Later, stained with fluorochrome-labelled Abs. After staining, cells were fixed by using 1% paraformaldehyde in FACS buffer. Cells were acquired in BD-FACS Aria III and BD-FACS Accuri (BD, Franklin Lakes, NJ). The analysis was performed using BD-FACS DIVA, BD-C6, and Flowjo software (BD, Franklin Lakes, NJ).
8
Macrophage Isolation and Characterization
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Macrophages were analyzed and sorted using a fluorescence-activated cell sorter (FACS) (BD Biosciences, San Jose, CA, USA) as previously described. Briefly, the AD tissues were dissected, carefully cut into small pieces, and enzymatically digested with collagenase II (1.5 mg/ml), elastase (0.25 mg/ml), and DNase I (0.5 mg/ml) for 1 h at 37°C. After digestion, the tissues were passed through 70-μm cell strainers. After washing, anti-CD16/32 antibody was used to block the non-specific binding. Fixable viability stain 510 (564406, BD Biosciences, San Jose, CA, USA) and the following antibodies were used for flow cytometry: CD45-APC-Cy7 (557659, BD Biosciences), CD11b-PE-Cy7 (552850, BD Biosciences), F4/80-BV421 (565411, BD Biosciences), CD86-PE (553692, BD Biosciences), and CD206-APC (565250, BD Biosciences). For flow cytometric sorting, cells were resuspended in the FACS buffer at 20 × 106 cells/ ml and separated on a MoFlo High-Performance Cell Sorter (Dako Cytomation, Carpinteria, CA, USA). The results were expressed as the absolute number of cells per mg of tissue. Data were analyzed with the FlowJo software (FlowJo LLC, Ashland, OR, USA).
9
Microglial Polarization Analysis in Brain Injury
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For flow cytometry analysis of microglial polarization status in the injured brain, the isolated microglia were stained with fluorescently labeled antibodies: CD11b-FITC (BD Biosciences), CD45-PE-Cy5 (BD Biosciences), CD163-APC (AbD Serotec), and CD86-PE (BD Biosciences) at 4 °C for 30 min. Flow cytometry was performed on a FACS VERSE apparatus (BD Bioscience) and obtained data were analyzed by Flow Jo software 7.6.1(Tree Star, USA).
10
Multiparametric Flow Cytometry Analysis
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Erythrocytes-depleted spleen cells, MLN cells and PECs were analyzed using a 6-color FACS, following a standard protocol (34 (link)). Cells were stained with different combinations of conjugated monoclonal antibodies CD3-FITC (Biolegend, San Diego, CA, USA), CD19-PEcy7, CD8-APCcy7, Sca-1-PE, CD25-APC, CD69-PEcy7, CD86-PE (BD Biosciences), CD11b-APCcy7, CD11c-APC, CD4-PE, F4/80-PEcy7, MHC II-APC (eBioscience, San Diego, CA, USA), and CD40-FITC (Southern Biotech). In the first panel, cells were immunphenotyped using mAbs to CD3, CD19, CD11b, CD11c, and Sca-1. A second panel was used to analyze T cell subsets and their activation status and consisted of mAbs to CD3, CD4, CD8, CD25, and CD69. In the third panel, myeloid cell activation status was analyzed using mAbs to CD11b, F4/80, CD40, CD86, and MHC class II. Non-viable cells that stained positive for 7-AAD (eBioscience), were excluded from the analysis. For each antibody, appropriate isotype control was used. Data were collected on 30,000 cells using BD FACS Canto II (BD Biosciences) and analyzed using BD FACSDiva software (BD).
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