Anti cd40 hb14

Cell surface markers expression on OECs, DCs, and CD4 T cells was analyzed by flow cytometry using the following antibodies and kits: anti-CD80 (L307.4), anti-CD83 (HB15e) and anti-CD86 (FUN-1), all from BD Pharmingen; anti-HLA-ABC (REA230), anti-HLA-DR (AC122), anti-CD83 (HB15), anti-CD40 (HB14) and anti-Cd11c (MJ4-27G12), all from Miltenyi-Biotech; anti-CD25 (BC96) and anti-CD69 (FN50) (eBioscience) and FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend), following the manufacturer's instructions. Briefly, cells were washed with 0.5% BSA/2 mM EDTA in PBS (staining buffer), Fc receptors blocked with human IgG serum (200 μg/ml)(Sigma-Merck) for 30 min at RT, and stained with the fluorescence-labeled antibodies. Cell markers expression was measured by flow cytometry (BD FACSCalibur) and data analysis was performed using FlowJo software (Tree Star).
IFNγ, TNFα, TGFβ, IL-1β, IL-2, IL-4, IL-6 and IL-10 (Invitrogen), and IL-8 and IL-12 (Inmunotools) were quantified in cell-free supernatants using specific solid phase sandwich ELISA cytokine kits following the manufacturer's instructions. The resulting color was measured (BioTek ELx800 Absorbance Microplate Reader).

Fresh PBMCs (10–20 × 10⁶ cells, final concentration 10 × 10⁶ cells/ml) were stimulated for 3 h at 37°C with 0.6 nmol/ml PepTivator C. albicans MP65 (pool of 15-amino-acid-length peptides with 11 amino acid overlap, Miltenyi Biotec) in RPMI + 5% human serum in the presence of 1 μg/ml anti-CD40 (HB14, Miltenyi Biotec). After stimulation, the PBMCs were labeled with PE-conjugated anti-CD154 (5C8, Miltenyi Biotec) and enriched with anti-PE magnetic beads (Miltenyi Biotec) (41 (link)). After enrichment, the cells were stained with PerCP-Cy5.5 anti-CD4 (RPA-T4, Biolegend), AlexaFluor700 anti-CD3 (SK7, Biolegend), and APC-Cy7 anti-CD45RA (HI100, Biolegend), and the antigen-specific T cells were gated as CD3⁺CD4⁺CD45RA⁻CD154⁺ single live cells for single cell sorting.