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Cell cultures PBMCs were isolated by Lymphoprep (Nycomed, Norway) density gradient separation and washed with RPMI-1640 (Gibco BRL, USA). Cells were suspended in RPMI-1640 with 10% fetal calf serum (Gibco BRL, USA) and 25 mM of Hepes (Gibco BRL) at a density of 2x106 cells/mL. Cells were stimulated in six-well culture plates (Becton Dickinson Labware, USA) in a volume of 5 mL with phorbol 12-myris-tate 13-acetate (PMA) (Sigma, USA) at a concentration of 10 ng/mL plus ionomycin (Sigma, USA) at a concentration of 2 pM for 4 h, 8 h and 24 h at 37°C in 5% carbon dioxide in an incubator. Cells cultured for 4 h without stimulation served as a control because, in a preliminary study of unstimulated cells, a longer incubation of up to 24 h showed no difference in the frequency of IL-10-producing T cells. Brefeldin A (Sigma, USA) at a concentration of 10 pg/mL was added during the last 2 h of incubation to inhibit cytokine secretion. After harvest, the cell viability was assessed by the trypan blue dye (Gibco BRL, USA) method. The cells were then centrifuged at 500 xg for 10 min and washed in phosphate buffered saline (PBS) with 0.2% fetal calf serum and 0.1% sodium azide (BDH Inc, Canada), referred to as washing buffer. The cells were transferred to 5 mL polystyrene tubes (Becton Dickinson Labware, USA) with 1x106 cells/tube and were suspended in washing buffer.