PBMCs from HLA-A*0201 positive healthy individuals were used for the differentiation and maturation of dendritic cells. PBMCs were seeded at a density of 5 × 106/well in a 24-well plate and after 2 h the non-adherent fraction (peripheral blood lymphocytes) was removed and cryopreserved for future incubation with mature autologous DCs. Differentiation of the adherent cells into dendritic cells was induced by GM-CSF (1000U/ml, #300-03, PeproTech, Rocky Hill, NJ, USA) and IL-4 (1000U/ ml; #200-04, PeproTech, Rocky Hill, NJ, USA) with replenishment every 2 days. On day 5, maturation of 0.5 × 106 DCs was induced using 100 ng/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) and maturation was checked on day 8 by flow cytometry (BD LSRFortessa X-20; Software Diva) using markers for CD83 (Anti-Human CD83 APC; #551073, BD Biosciences, Franklin Lakes, NJ, USA) & CD86 (Anti-Human CD86 FITC; #555,657, BD Biosciences, Franklin Lakes, NJ, USA).
Anti human cd86 fitc
Manufactured by BD
Sourced in United States
Anti-human CD86-FITC is a fluorescently labeled monoclonal antibody that binds to the CD86 surface marker expressed on antigen-presenting cells. It can be used for the identification and enumeration of CD86-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti human cd83 apc, by BD (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Anti human cd4 pe, by BD (1 mentions)
Anti human cd8 fitc, by BD (1 mentions)
Anti human cd3 percp, by BD (1 mentions)
Anti hla dr apc, by BD (1 mentions)
Anti human cd54 apc, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Annexin 5 fitc, by Thermo Fisher Scientific (1 mentions)
Cd80 fitc, by BD (1 mentions)
Cd86 pe, by BioLegend (1 mentions)
Cd83 fitc, by BioLegend (1 mentions)
7 aminoactinomycin d, by BioLegend (1 mentions)
Cd83 apc, by BioLegend (1 mentions)
Cd11c pe cy7, by BioLegend (1 mentions)
Cd14 apc, by BioLegend (1 mentions)
4 protocols using anti human cd86 fitc
1
Dendritic Cell Differentiation and Maturation
2
Immunophenotyping of Immune Cells
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For cell surface and intracellular staining, standard protocols were followed using monoclonal antibodies. Cells and antibodies were co-incubated on ice in the dark for 30 minutes. For intracellular staining of cytokines, cells were permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences, San Jose, CA, USA) for 30 minutes on ice, followed by an additional 30-minute incubation with antibodies in the dark at 4°C. The following antibodies were used: Anti-HLA-DR-APC (BD, cat. no.: 339194), anti-human CD54-APC (BD, cat. no.: 561899), anti-human CD86-FITC (BD, cat. no.: 560958), anti-human CD3-Percp (BD, cat. no.: 552851), anti-Human CD4-PE (BD, cat. no.: 557344), anti-Human CD8-FITC (BD, cat. no.: 555366), anti-human Ki-67-APC (BioLegend, cat. no.: 350514), and anti-IFN-gamma-APC (BD, cat. no.: IC285A-100). Imaging flow cytometry was performed using the ImageStreamX MarkII quantitative imaging flow cytometer, while conventional flow cytometry was performed using the BD FACS Calibur flow cytometer.
3
Immunophenotyping of Dendritic Cells
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Antibodies and other reagents used were fixable viability dye e-flour 450, Annexin V FITC (ebioscience); anti-human CD86-FITC, CD80-FITC, Propidium Iodide (PI) (BD Pharmingen, San Jose, CA); anti-human CD11c PE, HLA-DR PerCP, CD80-PE (Becton Dickinson, San Jose, CA); CD282-FITC, CD284-PE, Donkey anti-rabbit-FITC, CD86-PE, CD83-APC, CD11c-PE-Cy7, CD83-FITC, CD14-APC, and 7 aminoactinomycin D (7AAD (Biolegend, San Diego, CA). Data acquisition was on a Miltenyi MacsQuant flow cytometer (Miltenyi Biotech, San Diego, CA) on a log scale and analysis was performed with FlowJo 7.6.5 software (Tree Star, Ashland, OR).
4
Detailed Immunophenotyping of Dendritic Cells
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Primary unconjugated antibodies used in this study were anti-human CD1a clone OKT6 culture supernatant (from American Type Culture Collection), anti-human HLA-I clone W6/32, anti-human DC-SIGN (27 (link)) (a gift from Dr. Angel Corbí). ICAM-I IgG supernatant (HV5/3) and VCAM-I supernatant, a gift from Dr. Sánchez-Madrid. Fluorescein-coupled polyclonal goat anti-mouse IgG (H + L; Caltag Laboratories) was used as the secondary antibody in FACS analysis. The following conjugated antibodies were used: anti-human CD1d-PE, anti-human HLA-DR-PE, anti-human CD80-PE, anti-human CD86-FITC, anti-human CD25-APC, anti-human CD83-APC (all from BD Biosciences), anti-human iNKT-PE (Miltenyi Biotec), and anti-human CD8-PECy7 (Beckman Coulter), anti IFNγ-AF488 (BD Biosciences), anti FoxP3-AF488 (Biolegend). Flow cytometry was performed following standard protocols on a BD FACSCalibur cytometer. Graph bars summarizing flow cytometry data are expressed as relative mean fluorescence intensity (MFI), which is defined as the MFI of any sample divided by the MFI of the immature dendritic cells (iDCs).
For intracellular staining, cells were fixed with PBS-PFA 4% for 20 min, then, the cells were washed twice in PBS containing 0.1% saponin (Sigma-Aldrich), incubated with the desired antibodies in 100 ml of PBS containing 1% saponin for 30 min at room temperature, and washed with PBS/0.1% saponin buffer.
For intracellular staining, cells were fixed with PBS-PFA 4% for 20 min, then, the cells were washed twice in PBS containing 0.1% saponin (Sigma-Aldrich), incubated with the desired antibodies in 100 ml of PBS containing 1% saponin for 30 min at room temperature, and washed with PBS/0.1% saponin buffer.
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