Briefly, freshly isolated cells were incubated for 15 min at RT in the dark with 5 μm CFSE in PBS/0,1% BSA. Afterwards, excess dye was washed by centrifugation and cells were re-suspended in fresh complete medium and plated at a concentration of 1 × 106 cells per well in 96-well flat-bottom plates (Costar, Acton, MA, USA). Cells were stimulated with 10 μg/mL of complete somatic antigen (AgS) or with 5 μg/mL of antigenic fractions

(F9, F13, F17). The response was also measured when cells were costimulated with anti-CD3/CD28 antibodies; 0.5 μg/ml anti-CD3 (plate bound) and GDC-0973 solubility dmso 0.5 μg/mL anti-CD28 antibodies (BD Biosciences, Pharmingen, San Diego, CA, USA) were used in culture with antigen and fractions. After 72 h of culture cells were collected, washed with PBS, stained with anti-CD4-PerCP and anti-CD8-APC

antibodies (BD Biosciences, Pharmingen, San Diego, CA, USA) as described above and analysed in FACS. Cell proliferation, tracked by dye dilution, was monitored in CD4+ and CD8+ cells, and division index was calculated (DI = number of all cells/number of parent cells). The MLN cells at a concentration of 5 × 105 per well were plated onto 96-well flat-bottom plates (Costar) and treated simultaneously with 10 μg/mL of somatic antigen or with 5 μg/mL of separate antigenic fractions and 10 ng/mL of recombinant tumour necrosis factor-α (rTNF-α) or 100 nm synthetic glucocorticoid, dexamethasone, DEX (Sigma-Aldrich, Steinheim, Germany) NVP-LDE225 manufacturer in complete medium. Cells were cultured for 72 h, then collected and harvested by centrifugation at 800 g for 10 min. Cell pellets were resuspended in 100 μL of PBS (pH 7.4) with 2% BSA (Sigma-Aldrich). The effect of a stimulant dose on cell apoptosis was determined in a preliminary study using ssDNA ELISA. The method is based on the selective denaturation of DNA in apoptotic cells by formamide and detection of denatured DNA with a monoclonal antibody to single-stranded DNA (ssDNA).

The assay was performed following the manufacturer’s protocol using an ssDNA Apoptosis ELISA kit (Chemicon International, Inc., Temacula, CA, USA). Apoptosis Astemizole of specific populations of T cells was measured by four-colour flow cytometry. Cells were phenotyped for surface markers: 1 × 106 cells were incubated with 10 μL rat Alexa anti-mouse CD3, Allophycocyanin (APC) anti-mouse CD25, phycoeritrin (PE) anti-mouse CD4 (L3T4) and peridinin–chlorophyll–protein complex (PercP) anti-mouse CD8 monoclonal antibodies (BD Biosciences, Pharmingen) for 30 min at 4°C. The CD25 molecule (IL-2R-α chain p55) is widely used, but it is not a unique marker for Treg as it is also expressed on activated effector T cells. For this reason, we analysed CD4+CD25+ cells with a high expression of the IL-2Rα chain (CD4+CD25hi) to distinguish the natural from adaptive Treg subsets.