Anti human cd25 apc

PBMC from 20 healthy volunteers were isolated from whole peripheral blood using a density gradient (LymphoprepTM, AXIS-SHIELD) centrifugation and frozen until use. Briefly, PBMC were cultured for 3 days in RPMI 1640 media supplemented with 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin and 20% fetal calf serum (GibcoTM, Life Technologies). For activation, 5 μg/ml PHA (Sigma-Aldrich) and 50 U/ml of recombinant human IL-2 (Roche, Sigma-Aldrich) were added to the media. Viability staining (Live/Dead Fixable Dead Cell Stain kit, Invitrogen) was performed to gate on viable cells and T cell activation was assessed by staining for T cell markers (antibodies: anti-human CD3-APC-H7, CD4-PE-Cy7, and CD8-V500; BD Biosciences) and markers of T cell activation (antibodies: anti-human CD25-APC, HLA-DR-FITC and CD38-PerCP-Cy5.5; BD Biosciences). Cells were collected on an LSR II instrument (Becton Dickinson) and T cell activation markers analysis was performed using the FlowJo software.

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.