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3 protocols using non labeled abs

1

Murine Dendritic Cell Isolation and Stimulation

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Recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (rmIL)-4 were purchased from R&D Systems (Minneapolis, MN, USA), propidium iodide (PI), and ovalbumin (OVA) were purchased from Sigma-Aldrich (Steinheim, Germany), and lipopolysaccharide (LPS) and OVA-Alexa 488 were purchased from Invitrogen (Carlsbad, CA, USA). The following FITC- or PE-conjugated monoclonal antibodies (Abs) and non-labeled Abs were purchased from BD Biosciences (San Jose, CA, USA): FITC-annexin V, CD16/32 (2.4G2), CD11c (HL3), IA[b] (AF6–120.1), IFN-γ, CD4, PE-CD8, CD4. Cytokine ELISA primary and secondary -antibodies specific for murine IL-1β, IL-6, IL-12p70, IFN-γ, IL-2, IL-10, and TNF-α were purchased from BD Biosciences (San Jose, CA, USA). 5-Bromo-2′-Deoxy-Uridine Labeling and Detection Kit III and collagenase D were purchased from Roche (Salt Lake City, UT, USA).
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2

Murine Dendritic Cell Activation Assay

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Recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (rmIL)-4 were purchased from R&D Systems (Minneapolis, MN, USA), Dextran-FITC (40,000 Da), propidium iodide (PI), ovalbumin (OVA), and mitomycin C (MMC) were purchased from Sigma-Aldrich (Steinheim, Germany), and carboxyfluorescein succinimidyl ester (CFSE), lipopolysaccharide (LPS), and Pam3CSK4 (Pam3) were purchased from Invitrogen (Carlsbad, CA, USA). The following FITC- or PE- conjugated monoclonal antibodies (Abs) and non-labeled Abs were purchased from BD Biosciences (San Jose, CA, USA) : FITC-annexin V, CD16/32 (2.4G2), CD11c (HL3), CD40 (HM40–3), CD80 (16–10A1), CD86 (GL1), H-2Kb (AF6-88.5), I-A[b] (AF6–120.1), and CCR7 (4B12). Cytokine ELISA primary and secondary -antibodies specific for murine IL-1β, IL-6, IL-12p70, IFN-γ, IL-4 and TNF-α were purchased from BD Biosciences (San Jose, CA, USA), and the OT-1 peptide (OVA257-264) was purchased from Invivogen (San Diego, CA, USA). CD4+ T cell isolation kit II, CD11c Isolation Kit II and Separation Columns were purchased from MACS Miltenyi Biotec (Auburn, USA). 5-Bromo-2′-Deoxy-Uridine Labeling and Detection Kit III were purchased from Roche (Salt Lake City, UT, USA).
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3

Murine Lymphocyte Cytokine Profiling

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OVA and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (Steinheim, Germany). HPLC grade water and acetonitrile were purchased from Daejung Chemical (Siheung-si, Gyeonggi-do, Republic of Korea). Spin-X centrifuge filter 0.45 μm with cellulose acetate was purchased from Costar (Corning Incorporated, Corning, NY, USA). The following FITC- or PE-conjugated monoclonal anti bodies (Abs) and non-labeled Abs were purchased from BD Bioscience (San Joes, CA, USA): FITC- IFN-γ, PE-CD8, and CD16/32 (2.4G2). The cytokine ELISA primary and secondary–antibodies specific for murine IL-2, IFN-γ were purchased from BD Biosciences (San Jose, CA, USA). The 5-Bromo-2 –Deoxy-Uridine Labeling and Detection Kit III, and collagenase D was purchased from Roche (Salt Lake City, UT, USA).
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