Cultured PB CD14+ cells (purity of positive cells ranged between 72 and 90%) were resuspended in TRIzol® (Invitrogen, Life Technologies) total RNA isolation reagent (n = 4). As a positive control for the IDO mRNA expression PB CD14+ cells (0·25 × 106/well) were cultured in the presence of M-CSF (200 U/ml) for five days and on the fifth day IFN-γ (10 ng/ml) was added for 24 h . After the culture the cells were resuspended in Ultraspec (Biotecx, Houston, TX, USA) lysis buffer. RNA isolation was performed as described by the manufacturer. Oligo-p(dT)-primed cDNA were synthesized using avian myeloblastosis virus (AMV) reverse transcriptase in a 20 µl reaction volume (1st Strand cDNA Synthesis Kit for RT-PCR, Roche Diagnostics, Mannheim, Germany). To exclude possible contamination of genomic DNA, AMV reverse transcriptase was omitted from the control RT reactions. Each reaction mixture was incubated at 42°C for 1 h using a DNA thermal cycler (Perkin-Elmer, Norwalk, CT, USA). A heat inactivation step at 99°C for 5 min was performed to denaturate reverse transcriptase, and this was followed by a cooling step to +4°C for 5 min. The reagents and capillaries for Real-Time quantitative PCR (LightCycler™, Roche Molecular Diagnostics, Mannheim, Germany) were purchased from the Roche Molecular Diagnostics. Real-time RT-PCR for the quantification of IDO expression was conducted using Light Cycler – FastStart DNA Master SYBR Green I kit (Roche) according to the manufacturer's instructions. Additional MgCl2 to achieve the optimal final concentration 4 mm for IDO and 2 mm for β-actin, each primer at 10 pmol/µl and 2 µl of 1 : 10 and 1 : 100 diluted template cDNA was used. The specific primers used for IDO were: sense (5′-AACTCCTGGACAATCAG TAAAG-3′) and antisense (5′-ATATATGCGAAGAACACT GAAAAA-3′). As a control constitutively expressed β-actin gene was amplified from the same pool of cDNA to normalize the concentration of cDNA; sense primer (5′-AGCCTCGCCTTTGC CGA- 3′) and antisense (5′-CTGGTGCCTGGGGCG-3′) . At the beginning of thermal cycler program activation of the FastStart Taq DNA Polymerase at 95°C for 10 min was done. PCR amplification steps for IDO (15 s at 95°C, 5 s at 60°C, 28 s at 72°C) and for β-actin (15 s at 95°C, 5 s at 67°C, 9 s at 72°C) were repeated 50 times. Fluorescence was measured at channel F1 after each elongation. Melting curves were done by lowering the temperature to 65°C, then raising the temperature by 0·1°C/ s to 95°C for IDO and to 99°C for β-actin and measuring the fluorescence continuously. The standard curve for IDO was constructed using IDO positive cDNA in four concentrations (1 : 10, 1 : 100, 1 : 1000, 1 : 10 000) and was used for calculations. Finally, the products were analysed by electrophoretic separation on a SeaKem 1·5% agarose gel (FMC Bioproducts, Rockland, ME, USA).