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3 protocols using hla dr ln3

1

Phenotypic Characterization of Immune Cells

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Monocyte purity, proper Mo-DC differentiation and the basal state of activation of both cells were assessed by flow cytometry using the following anti-human monoclonal antibodies: CD14 (M5E2, Biolegend, San Diego, CA, USA), CD1b (SN13, Biolegend), CD1d (51.1, Biolegend), CD11c (3.9, eBioscience), CD80 (2D10, Biolegend), HLA-DR (LN3, eBioscience).
iNKT and T cell determinations were performed in total PBMCs or in CD14 fractions, using CD1d-PBS57 tetramers (NIH Tetramer Core Facility, Emory University, Atlanta, GA, USA) and the following anti-human monoclonal antibodies: CD3 (OKT3, eBioscience), CD4 (OKT4, Biolegend), and CD8 (RPA-T8, eBioscience). The purity of T cell clones VM-D5 and JS63 was assessed by using CD1d-PBS57 tetramers (NIH Tetramer Core Facility, Emory University, Atlanta, GA, USA) together with anti-human CD3 (OKT3, eBioscience) monoclonal antibody. The purity of T cell clones s33d, GG33A, and DS1C9b was assessed by using anti-human TCR Vβ13.1 (IMMV222), Vβ18 (BA62.6), and Vβ7.1 (ZOE), monoclonal antibodies from Immunotec (Immunotec Research Inc, Canada). Cells were acquired in a FACS Canto II (BD Biosciences, San Diego, CA, USA) using the BD FACSDiva™ software (BD Biosciences). Data analysis was performed with FlowJo® v10 (FlowJo LLC, Ashland, OR, USA).
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2

In Vitro Generation of Human Monocyte-Derived Dendritic Cells

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Human MO-DC were generated in vitro as previously described (14 ). Briefly, CD14+ monocytes were magnetically isolated from PBMC (Miltenyi) and cultured in RPMI containing 10% FCS supplemented with GM-CSF (100ng/ml) and IL-4 (20ng/ml) (R&D Systems). Cultures were fed on day 4. On day 8, DC were seeded into poly(2-hydroxyethyl-methacrylate) (Sigma) coated 12-well culture plates (Corning Costar) at a density of 1 million cells/well and stimulated for 2 days with a cocktail containing IL-6 (10ng/ml), PGE2 (0.1μM) along with IL-1β (10ng/ml), IL-36α, IL-36β or IL-36γ (100ng/ml) in 500μl complete RPMI. DC phenotype was analyzed by flow cytometry as described above using antibodies against CD86 (FUN-1, BD), HLA-DR (LN3, eBioscience), CD1a (HI149, eBioscience), CD11c (3.9, eBioscience), CD123 (6H6, Biolegend) and appropriate isotype control antibodies.
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3

Phenotypic Analysis of Immune Cells

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For phenotypic analysis, cells were labelled with Abs recognizing CD11c (3.9), CD40 (5C3), CD45 (HI30), CD83 (HB15e), HLA-DR (LN3, all eBioscience), BDCA1/CD1c (AD5-8E7, Miltenyi Biotec), BDCA3/CD141 (AD5-14H12, Miltenyi Biotec), CD86 (2331, BD Horizon), CLEC9A (8F9, BioLegend), HBsAg (Acris), a lineage cocktail including CD3 (UCHT1, eBioscience), CD14 (61D3, eBioscience), CD19 (HIB19, eBioscience) and CD56 (MY31, BD Biosciences), and the live/dead marker Aqua (LifeTechnologies). Fluorescence was measured using a FACS Canto II (BD Biosciences).
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