11 Subsequent experiments, involving TG and TT only, were perform

11 Subsequent experiments, involving TG and TT only, were performed in RPMI-1640 medium (Gibco BRL, Life Technologies, Taastrup, DK) containing penicillin/streptomycin and supplemented with l-glutamine (2 mm). All culture experiments were performed in the presence of 30% autologous serum. 5-Carboxy-2′,

7′-dichlorofluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR), kept as a stock solution of 5 mm in dimethylsulphoxide, was diluted to 50 μm in α-MEM before use. The CFSE was added to suspensions of PBMC in α-MEM to a final concentration of 2 μm. The cells were incubated with CFSE for 10 min in a humidified incubator at 37°, 5% CO2. Flow cytometry was performed using BD Biosciences FACScan or FACSCalibur flow cytometers with argon laser KU-60019 excitation (488 nm) and the data were analysed using CellQuest software. The signals from CFSE and PerCP-anti-CD4 (or anti-CD14) were detected buy SCH 900776 in channels FL1 and FL3, respectively. CD4+ T cells, or monocytes, were identified using a combination of forward scatter versus side scatter gating and the appropriate PerCP-conjugated marker. In all measurements, the background proliferation was subtracted from the proportion of dividing cells

upon antigen stimulation. The CFSE-labelled PBMC were distributed in 96-well culture plates (5 × 105 cells/well) and incubated with TT (10 μg/ml), TG (30 μg/ml), KLH (30 μg/ml) or without antigen in α-MEM containing 30% autologous serum (total volume = 200 μl). Following culture in a humidified incubator Fossariinae at 37°, 5% CO2 for 1, 5, 7 or 9 days, the supernatants were harvested for cytokine analysis and the cells were washed in PBS (4 ml) and stained with

PerCP-anti-CD4 for assessment of CD4+ T-cell proliferation by flow cytometry. Proliferation was expressed as % dividing cells, determined as 100 × (no. of CD4+ T cells displaying CFSE fluorescence < 50% the fluorescence signal for non-dividing cells)/(total no. of CD4+ T cells). The content of IL-2, IFN-γ, IL-4, IL-5, TNF-α and IL-10 in culture supernatants at days 1, 5, 7 and 9 was quantified by means of a Th1/Th2 Cytometric Bead Array kit using a FACSCalibur flow cytometer. Data analysis was performed using the Cytometric Bead Array software (BD Biosciences). Interleukin-10 secretion by individual cells was examined with a cytokine capture assay using anti-IL-10 and anti-CD45 co-conjugated beads (MACS Miltenyi, Biotech Line AS, Slangerup, Denmark). Lymphoprep-purified PBMC were suspended at a density of 106 cells/ml in RPMI-1640 with 30% autologous serum and challenged with TG (30 μg/ml), TT (10 μg/ml) or no antigen over night at 37°. The IL-10 secretion assay was performed, without erythrolysis, according to the manufacturer’s protocol.

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