Culture medium was refreshed twice weekly. At subconfluency, MSC were removed from culture flasks using 0·05% trypsin–ethylenediamine tetraacetic acid (EDTA) (Life Technologies) and reseeded at 1000 cells/cm2. MSC were characterized by means of

immunophenotyping and by their ability to differentiate into adipocytes and osteoblasts. MSC cultured between two to six passages were used. MSC from these passages did not differ in their ability to differentiate or to exert their immunosuppressive functions. Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy blood donors (Sanquin, Rotterdam, the Netherlands) Selleckchem BMN673 by density gradient centrifugation using Ficoll-Paque PLUS (density 1·077 g/ml; GE Healthcare, Uppsala, Sweden). Cells were frozen at −150°C until further use in RPMI-1640 medium with GlutaMAXTM-I (Life Technologies) supplemented with 1% P/S, 10% human serum (Sanquin) and 10% dimethylsulphoxide (DMSO; Merck, Hohenbrunn, Germany). Mixed lymphocyte reactions (MLR) were set up with 5 × 104 effector PBMC and 5 × 104 γ-irradiated (40 Gy) allogeneic PBMC in round-bottomed 96-well plates (Nunc, Roskilde, Denmark). MLR were cultured in MEM-α supplemented with 2 mM L-glutamine, 1% P/S and 10% heat-inactivated human serum for 7 days in a humidified

atmosphere with 5% CO2 at 37°C. Effector–stimulator cell combinations were chosen on the basis of a minimum of four human leucocyte antigen (HLA) mismatches. The immunomodulatory capacities of MSC and belatacept (Bristol-Myers-Squibb, New York, NY, USA) on MLR were determined in suppression assays. For learn more flow cytometric analysis, effector PBMC were labelled with BD Horizon violet cell proliferation dye 450 (VPD450; BD Biosciences, San Jose, CA,

USA). For distinction from effector PBMC, γ-irradiated allogeneic stimulator PBMC (40 Gy) were labelled using the PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich). When cell proliferation was assessed by thymidine incorporation, [3H]-thymidine (0·25 μCi/well; PerkinElmer, Groningen, the Netherlands) was added on PAK6 day 7, incubated for 8 h and its incorporation was measured using the Wallac 1450 MicroBeta TriLux (PerkinElmer). PBMC were stained with monoclonal antibodies (mAbs) against CD3 (AmCyan), CD4 [allophycocyanin (APC)], CD8 [fluorescein isothiocyanate (FITC)], CD28 [peridinin chlorophyll-cyanin 5·5 (PerCP-Cy5·5)] and either CD3+CD8+CD28− cells and CD3+CD4+ cells or CD3+CD28− cells and CD3+CD28+ cells were sorted on the BD FACSAria II cell sorter (BD Biosciences). Effector populations for MLR consisted either of CD3+CD28− cells only (mean purity 97·8%, range 96·3–98·8%), CD3+CD28+ cells only (mean purity 96·2%, range 93·0–99·5%) or a combination of 10% CD3+CD8+CD28− cells (mean purity 92·3%, range 88·4–94·72%) and 90% CD3+CD4+ cells to provide help (mean purity 98·2%, range 97·2–99·5%).