PEM (detached by 15 min incubation at 37°C with 15 mM lidocaine (Sigma-Aldrich) plus 5 mM EDTA in PBS), purified BM-DC or unfractionated splenic cells (2 × 105 leukocytes) were pre-incubated for 30 min on ice in 0.1 ml of FCS-RPMI containing 40% mouse serum and 50 μg/ml of non-immune mouse IgG2a (BD Biosciences), in order to decrease non-specific binding. Subsequently, 0.1 ml solutions of receptor-specific or control mAb were added, to give final mAb concentrations of 10 μg/ml, and the incubation was continued for 50 min. Unbound mAb were removed by washing with 2 ml of ice-cold PBS, and the cells were incubated for another 50 min with 5 μg/ml of PE-conjugated secondary Ab in 0.2 ml FCS-RPMI. In order to enable identification of DC and macrophages, splenic cells were subjected to additional incubation with 5 μg/ml of allophycocyanin-conjugated anti-CD11c (clone N418) or F4/80 mAb (eBioscience), respectively. Following washing twice, binding of fluorescently-labelled Ab to cells was assessed by flow cytometry.
The surface expression of MHC-II, CD40 and CD86 was also determined by flow cytometry, by direct labelling of these molecules with 5 μg/ml of PE-conjugated MHC-II-, CD40-, or CD86-specific or isotype-matched control mAb obtained from BD Biosciences or eBioscience.
The surface expression of MHC-II, CD40 and CD86 was also determined by flow cytometry, by direct labelling of these molecules with 5 μg/ml of PE-conjugated MHC-II-, CD40-, or CD86-specific or isotype-matched control mAb obtained from BD Biosciences or eBioscience.