DCs were blocked with fetal bovine serum (FBS) 20% for 2 h. For indirect staining of Pgp in mDCs, 0·5 × 106 DCs were incubated overnight at 4°C with the primary anti-Pgp PD-0332991 manufacturer monoclonal antibody (mAb) JSB1 (1/50 with
FBS 10%), anti-MRP1 mAb (4124) and DC LAMP antibody (1/50 with FBS 10%). Before incubation, cells were permeabilized to anti-Pgp mAb JSB1 incubation. After incubation, cells remained for 30 min at room temperature. The DCs were then incubated with the secondary antibodies Alexa 647 and Alexa 488 (1/100 with FBS 1%) for 45 min and washed. Finally, DCs were mounted in DAPI. Analysis of cell surface marker expression was performed using the dual-colour inmunofluorescence technique (Leica TCS-SL confocal espectral microscope, Mannheim, Germany) equipped with image analysis software (Leica confocal software). Distinguishing DCs from monocytes was also defined functionally by the ability to stimulate an allogeneic mixed leucocyte reaction (MLR) [20, 21]. Thus, we tested not only phenotypical changes, but also functionally tested CD3 proliferation. We performed a CFSE study to analyse the effector function of these DCs; the results supported the phenotypical changes and also emphasized the distinction from macrophages. Lymphocytes were stained with CFSE and exposed selleck chemicals llc to mDCs (under hypoxia or LPS stimuli) with or without ABC transporter inhibitors. After 24 h,
medium was removed and co-culture was performed with fresh medium. Allogeneic CFSE-labelled PBMCs (2 × 105) were cultured alone (negative control) or in the presence of DCs collected aminophylline at the end of the 7-day culture after stimuli exposure (DC : T cell ratio 1:10; final volume 200 μl RPMI 10% FBS).
As positive control responder, PBMCs were stimulated with 1 μg/ml (PHA). After 5 days of culture (37°C, 5% CO2) the proliferation of responder cells was determined by flow cytometry after labelling with CD20, CD4 and CD8 antibody to exclude DCs and to define different B and T lymphocyte subpopulations. No ABC transporter inhibitors were used in T and DC co-cultures. In addition, MLR with purified T and B cells was performed with the RosetteSep human T cell enrichment cocktail and the RosetteSep human B cell enrichment cocktail, respectively (Stemcell Technology, Grenoble, France) After cell isolation the MLR technique was carried out as described. Flow cytometry analysis was performed using FACS Canto and diva software (Becton Dickinson). Interleukin-2, -4, -6, -10, -17a, TNF-α and interferon (IFN)-γ secretion protein levels from cell supernatant were measured quantitatively following cell stimulation by CBA (BD Biosciences). Cytokine quantification was performed on stimulated and non-stimulated, and treated and non-treated (with ABC inhibitors) DCs, and on lymphocytes after MLR. Each experiment was performed at least three times and representative data are shown.