DC maturation after pulsation with HHP-killed cancer cells alone or after pulsation with HHP-killed cancer cells and poly(I:C) (DC-based HHP lung cancer vaccine) was evaluated by flow cytometry. DC phenotype was determined by staining with CD80-FITC, CD86-PE, CD83-PE-Cy5.5 (Beckman Coulter), CCR7 (CD197)-APC-eFluor780 (eBioscience), CD11c-APC (Exbio) and HLA-DR-PE-Cy7 (BD Biosciences) for 20 min. Cells viability was detected with DAPI staining. CD11c+DAPI negative DC were analyzed for mean fluorescence intensity (MFI) of the particular maturation marker. To determine cytokine production, cell supernatants were harvested 24 h after DC’s stimulation and stored at -80°C. IL-12p70, IL-10, IFN-α and TNF-α were determined using Luminex assay (MILLIPLEX™ Human Cytokine/Chemokine Kit, Merck Millipore) by Luminex 2000 (Luminex).
Cd86 pe
Manufactured by Beckman Coulter
Sourced in United States
CD86-PE is a flow cytometry reagent used to detect and quantify CD86 expression on the surface of cells. CD86 is a co-stimulatory molecule that plays a role in the activation of T cells. The PE (Phycoerythrin) fluorescent label allows for the visualization and analysis of CD86-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd80 fitc, by Beckman Coulter (3 mentions)
Summit 5, by Beckman Coulter (2 mentions)
Anti cd56 pe, by Beckman Coulter (2 mentions)
Anti cd3 pc7, by Beckman Coulter (2 mentions)
Cd83 conjugated to apc, by BioLegend (2 mentions)
Anti cd4 pc5 anti cd8 apc, by Beckman Coulter (2 mentions)
Hla dr ecd, by Beckman Coulter (2 mentions)
Cd40 pe, by Beckman Coulter (2 mentions)
Anti cd25 ecd, by Beckman Coulter (2 mentions)
Anti foxp3 pe, by BioLegend (2 mentions)
Cd83 fitc, by Beckman Coulter (2 mentions)
Hla dr pe cy7, by BD (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Cd11c apc, by Exbio (1 mentions)
Milliplex human cytokine chemokine kit, by Merck Group (1 mentions)
Luminex 2000, by DiaSorin (1 mentions)
Cd4 fitc, by Beckman Coulter (1 mentions)
Cd27 pc7, by Beckman Coulter (1 mentions)
Cd3 ecd, by Beckman Coulter (1 mentions)
Fitc cd38, by Beckman Coulter (1 mentions)
7 protocols using cd86 pe
1
Evaluating DC Maturation in a Lung Cancer Vaccine
2
Profiling Gut-Associated Immune Cells
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PBMCs from UC patients and healthy controls were harvested and stained in duplicate for CD45-PC7, CD3-ECD, CD8-PE, CD4-FITC, CD19-PC5, CD38-FITC, CD27-PC7, CD86-PE (Beckman Coulter, Brea, CA), CXCR5-PC5 and ICOS-PE (eBioscience, San Diego, CA) at room temperature for 30 min. After being washed with PBS, the cells were characterized by flowcytometry analysis using the Beckman Coulter Cytomics FC500 (Beckman Coulter). Cells stained with separate antibodies were defined as certain TFH cells (CD4+ CXCR5+ TH cells and CD4+CXCR5+ICOS+ TH cells) and subsets of B cells (CD38+CD19+ B cells, CD86+CD19+ B cells and CD27+CD19+ B cells). Data were analysed with a CXP Analysis Cytometer (Beckman Coulter).
3
Phenotyping Dendritic and Proliferating Cells
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For phenotypic analysis of dendritic cells: Fluorochrome conjugated antibodies CD14-PC5, CD80-FITC, HLADR-ECD, CD40-PE and CD86-PE conjugated were purchased from Beckman Coulter Inc (Carlsbad, CA, USA). The antibody against maturation marker CD83-conjugated to APC was purchased from Bio Legend (San Diego, CA, USA).
For phenotyping proliferating PBMCs, anti-CD56-PE, anti-CD4-PC5 anti-CD8-APC, anti- CD25-ECD and anti-CD3-PC7 antibodies were purchased from Beckman Coulter Inc (Carlsbad, CA, USA). Anti-FOXP3-PE was purchased from BioLegendInc (San Diego, CA, USA). Flow cytometry was performed as described previously [27 (link)]. All the samples were acquired using MoFlo XDP flow cytometer configured with three different lasers-blue (488 nm), violet (405 nm) red (640 nm) analyzed using Summit 5.2 software (both Beckman Coulter Inc, Carlsbad, CA, USA).
For phenotyping proliferating PBMCs, anti-CD56-PE, anti-CD4-PC5 anti-CD8-APC, anti- CD25-ECD and anti-CD3-PC7 antibodies were purchased from Beckman Coulter Inc (Carlsbad, CA, USA). Anti-FOXP3-PE was purchased from BioLegendInc (San Diego, CA, USA). Flow cytometry was performed as described previously [27 (link)]. All the samples were acquired using MoFlo XDP flow cytometer configured with three different lasers-blue (488 nm), violet (405 nm) red (640 nm) analyzed using Summit 5.2 software (both Beckman Coulter Inc, Carlsbad, CA, USA).
4
Blocking and Staining of Immune Cells
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In order to prevent unspecific binding of antibodies a 15 min blocking step with 100% FBS (heat inactivated, Gibco, Life Technologies, Zug, Switzerland) was performed before staining. Staining was done at optimally titrated concentrations with mouse anti-human antibodies specific for surface markers of interest - CD83-FITC, CD86-PE, CD80-PC7, CD14-ECD, CD11c-PC5 (all purchased from Beckman Coulter, Nyon, Switzerland) - and with corresponding isotype control antibodies for 20 min at 4°C in the dark. In order to remove unbound antibodies, cells were washed once with FACS buffer (PBS+1.0% FBS+0.05% sodium azide) before being finally re-suspended in FACS buffer containing fixative (Beckman Coulter, Nyon, Switzerland). Fixed cells were stored at 4°C in the dark until measurement.
5
Flow Cytometry Analysis of Dendritic Cells
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The matured DCs were harvested and transferred to a V-bottom 96-well plate (Nalgene). The cells were pelleted, washed with ice-cold PBS supplemented with 2 mM EDTA (PBS/EDTA), and stained with the following fluorescent-labeled antibodies: CD83-FITC, CD86-PE (Beckman Coulter, Brea, CA, USA), or HLA-DR-Pe-Cy7 (Becton Dickinson, Franklin Lakes, NJ, USA). After 30–60 min incubation at 4 °C in the dark, the cells were pelleted and rinsed twice with ice-cold PBS/EDTA. The cells were resuspended, transferred to 5-mL round-bottom FACS tubes (Nalgene), supplemented with 100 ng/mL of DAPI (Thermo Scientific), and immediately analyzed using FACSAria II or a BD LSRFortessa flow cytometer (Becton Dickinson). The FlowJo software (Tree Star, Ashland, OR, USA) was used to analyze the acquired flow cytometry data.
6
Phenotypic Analysis of Dendritic Cells
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For phenotypic analysis of dendritic cells: Fluorochrome conjugated antibodies CD14-PC5 (CD-cluster of differentiation PC5-PE-Phycoerythrin cy5-cyanine, CD80-FITC, HLADR-ECD, CD40-PE and CD86-PE were purchased from Beckman Coulter Inc (CA, USA). The antibody against maturation marker CD83conjugated to APC was purchased from Bio Legend (San Diego, CA, USA) For phenotyping proliferating PBMCs, anti-CD56-PE, anti-CD4-PC5 anti-CD8-APC, anti-CD25-ECD and anti-CD3-PC7 antibodies were purchased from Beckman Coulter Inc. Anti-FOXP3-PE was purchased from BioLegend Inc. Flow cytometry was performed as described previously [25] . All the samples were acquired using MoFlo XDP ow cytometer con gured with three different lasers -blue (488nm), violet (405nm) red (640nm) analyzed using Summit 5.2 software (both Beckman Coulter Inc).
7
Immune Cell Profiling in Leukapheresis
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The quantity and percentage of various immune cell types in leukapheresis product and elutriation fractions were analyzed by an ABX Pentra 60 hematology analyzer (HORIBA). For other experiments, FACS analysis (Accuri C6 Plus; Beckman Coulter Life Sciences) was used. Monocytes were enumerated by CD45 + /CD14 + and propidium iodideÀnegative population (Beckman Coulter Life Sciences). Dendritic cells were gated by forward scatter and side scatter on the basis of cell size and cellular complexity, and further gated by anti-CD45 and propidium iodide. Phenotypic analyses of dendritic cells were performed using anti-CD209-APC, CD80-PE, CD83-PE, CD86-PE, CD11c-PE, HLA-DR-APC, HLA-ABC-APC and CCR7-APC (Beckman Coulter Life Sciences). To analyze cell type compositions in CUD-002 product, anti-CD45-APC, CD19-PE, CD20-PE, CD3-Percp-Cy5.5, CD56-PE, CD34-PE, lineage cocktail (CD3/CD14/CD16/CD19/CD20/CD56)-FITC, CD13-PE, CD33-PE and S100A9-PE (Beckman Coulter Life Sciences) were used.
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