Samples were run at 37°C on the BD™ LSR cytometer (BD Biosciences), and changes in FL5-H/FL4-H ratio were recorded for a total of 512 s (the basal line was recorded for 30 s before the cross-linking Ab was added). Isotype-matched mAb MOPC-21 was used in the assay as a negative control. Data analysis was done using the FlowJo software (Three Star). To analyze the respiratory burst kinetics, production of O was assayed by detection of reduced cytochrome c by freshly isolated monocytes as previously described 40. Briefly, cells were resuspended
in HBSS buffer supplemented with 10% FBS, 0.5 mM Ca2+ and 1 mg/mL glucose and plated over the coated mAb IWR1 at 1.5×105/100 μL in 96-wells plate. After 15 min of incubation at 37°C in 5% CO2 atmosphere, 80 μM cytochrome c (Sigma Aldrich) was added and the plate was kept at 37°C in VersaMax™ microplate reader (Molecular Devices,
Sunnyvale, CA, USA). Absorbance was measured at 550 and 468 nm during 3 h in 10-min intervals. Supernatants of cells (1×106/mL) stimulated either with plate-coated mAb, www.selleckchem.com/products/ABT-263.html ultra pure E. coli LPS or recombinant human M-CSF (rhM-CSF, ImmunoTools GmbH) for 24 h were collected and frozen at −20°C until required. Supernatants were analyzed by ELISA for IL-6, IL-8/CXCL8, IL-10, TNF-α (all from ImmunoTools GmbH) and IL-12p70 (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Freshly isolated cells were stimulated Sirolimus cost with plate-coated mAb or medium alone in a 24- or 48-well plate (Corning, Corning, NY, USA). Ultra pure E. coli LPS at 100 ng/mL or rhM-CSF (ImmunoTools GmbH) at 10 ng/mL were used as positive controls. After 24 (mDC) or 48 h (monocytes) of incubation, cells were harvested and apoptotic cells were detected by labeling with Annexin-V-FLUOS (Roche Applied Sciences, Penzberg, Germany) followed by flow cytometry analysis. mDC were visualized using an inverted Leica SP2 Confocal
microscope (Leica Microsystems, Wetzlar, Germany) under the 63×/1.32 oil Ph3 CS objective; final total magnification ×200. CbT were obtained from umbilical cord blood samples supplied by Cord Bank of Barcelona, according to guidelines approved by Ethical Committee with donor consent. Cord blood mononuclear cells were separated by Ficoll-Paque PLUS centrifugation (GE Healthcare Bio-Sciences AB) and CbT cells were purified by negative selection using the RosetteSep™ human T-cell enrichment cocktail (StemCell Technologies) that contained anti-CD16, anti-CD19, anti-CD36, anti-CD56, anti-CD66b and anti-glycophorin A mAb. Purity of the cell preparation was assessed by FACS using CD3 and CD45RA markers. In each experiment, >80% of the cells were CD3+CD45RA+. CFSE labeling of CbT cells was performed as previously described 41.