Apc anti human hla dr

The activation markers expression on resting memory CD4⁺ T cells were detected after IP-10 or CD3/CD28 stimulated and measured using the following fluorophore-conjugated antibodies: phycoerythrin (PE)-Cy7 anti-human CD3, allophycocyanin (APC)-Cy7 anti-human CD4, peridinin-chlorophyll-protein anti-human CD197/CCR7, fluorescein isothiocyanate (FITC) anti-human CD45RA, APC anti-human CD69, APC anti-human CD25, APC anti-human HLA-DR, APC mouse IgG1 κ isotype control, and PE mouse IgG1 κ isotype control (all from Biolegend). Samples were analyzed using an LSR II flow cytometer (BD Biosciences), which was adjusted with 10-peak color rainbow beads (Spherotech, Lake Forest, IL, USA). Gates were defined using appropriate isotype controls. Expression levels in each sample were analyzed with FlowJo v10 software (Ashland, OR, USA).

Perpheral blood mononuclear cells (PBMCs) were isolated from leukocyte cones (NC24) obtained from healthy donors supplied by the NHS blood and transplant service (UK) by Ficoll (GE Healthcare, US) gradient centrifugation (according to MACS Miltenyi Biotec protocol). CD14+ monocytes were seperated from PBMCs via magnetic positive selection using CD14+ beads and MACS column (Miltenyi Biotec, Germany). Immature dendritic cells (iDC) were culture in RPMI medium supplemented with FBS, interleukin-4 (IL-4, 500 U/ml; PeproTech, US) and granulocyte-macrophage colony-stimulating factor (GM-CSF, 800 U/ml; PeproTech) for 5 days. Next, the co-culture of iDC and Z_HER2:2395-IR700-treated SKOV-3 cells (ratio 1:2 iDC/cancer cell) was irradiated (16 J/cm²). DC maturation was induced by stimulating the iDC with E.coli lipopolysaccharide (LPS, 100 ng/ml; Sigma) for 12 h (a positive control). After 48 h, all floating cells were collected and with Pacific Blue™ anti-human CD14 (#367121), FITC anti-human CD86 (#374204) and APC anti-human HLA-DR (#307610) (all antibodies from BioLegend, US) and PI. DC maturation was evaluated on single, live (PI-) and CD14- gated cells populations by flow cytometry. Cell supernatants were collected at different time points and used for anti-IL6, anti-IL-10 and anti-TNF-α ELISA assays according to manufacture protocols (BioLegend, US).