T cell proliferation: Heparin anticoagulated blood (50 ml) was obtained from 10 randomly selected members of each of the three subject groups and centrifuged at 850 g for

20 min. Plasma was removed, and cells were suspended PF-02341066 cost in D-Hanks solution. This was layered onto Ficoll separation medium in a tube followed by centrifugation at 850 g for 20 min. Cells in the middle layer were carefully collected, which were peripheral blood mononuclear cells (PBMCs). PBMCs were washed 3 times in RPMI-1640 by centrifugation at 450 g for 10 min and then re-suspended in RPMI-1640 to a density of 1 × 108/ml. A fraction of this cell suspension was loaded onto a prewarmed Nylon Fiber column T (37 °C) with RPMI-1640 medium containing 10% FBS; the volume of the cell suspension was one-third that of the column. After sealing,

EX 527 mw the column was kept warm at 37 °C for 1 h, after which prewarmed RPMI-1640 (37 °C) was added at a flow rate of 3–4 ml/min. The opaque medium was collected, which contained T lymphocytes. T lymphocytes were re-suspended in RPMI-1640 containing 10% FBS at a density of 1 × 106/ml. Cell suspensions were added to a 96-well plate (100 μl/well) followed by adding PHA (final concentration: 20 μg/ml; and final volume in each well: 200 μl). As controls, cells without PHA were also included, and three wells were included for each group. Plates were incubated at 37 °C in a 5% CO2 atmosphere for 48 h. At 4 h before the end of incubation, MTT (20 μl; 5 g/l) was added and incubation was continued at 37 °C for the remaining 4 h. The plate was centrifuged, the supernatant was removed, and DMSO (100 μl/well) was added to dissolve the crystals followed by incubation for 15 min. Optical

density (OD) was measured with a new microplate reader (detection wavelength: 570 nm; reference wavelength: 630 nm), and a stimulation index (SI) was calculated: SI = ODexperiment/ODcontrol. Cytokine-induced killer (CIK) cell culture and assessment of tumouricidal activity: PBMCs were suspended in RPMI-1640 at a density of 1 × 106/ml. On day 0, γ-INF (1000 U/ml) was added followed by incubation at 37 °C in a 5% CO2 atmosphere for 24 h. On day 1, IL-1 (100 U/ml), CD3 mAb (50 ng/ml) and IL-2 (500 U/ml) were added followed by further incubation; half of the medium was refreshed every 3 day during which IL-2 was added. On day 6, CD3 mAb (50 ng/ml) was added again. On day 15, cells (CIK cells) were harvested and re-suspended in RPMI-1640 at a density of 1 × 106/ml; these were used as effector cells. K562 cells were used as target cells. Effector cells were mixed with target cells at a ratio of 10:1 and then added to a 96-well plate. As controls, effector cells or target cells alone were also added to three wells for each group. MTT (20 μl; 5 g/l) was added, and plates were incubated at 37 °C in a 5% CO2 atmosphere for 4 h followed by centrifugation.