Methods. Heparinized peripheral blood is stimulated with 1 ml of complete Roswell Park Memorial Institute (RPMI) 1640 medium containing 100 ng/ml phorbol 12-myristate 13-acetate (PMA) and 5 mcg/ml ionomycin (Sigma-Aldrich Corp., St.Louis, MO, USA) for 3 hours at 37°C. Following ex vivo immune activation, after red cell lysis with ACK buffer (0.15 M NH4CL, 1.0 mM KHCO3), leukocytes are lysed with 400 mcl of MagNA-Pure lysis buffer supplemented with an additional 1% (W/v) of dithiothritol (RAS, Mannheim, Germany), and the sample is frozen at -70°C. After thawing, mRNA is isolated with the RNA Blood mini Kit (Quiagen) device using the mRNA standard protocol for cells. RNA is reverse transcribed using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, life technologies). The 3 NFAT-regulated genes (IFN-γ, IL-2, GM-CSF) were identified as suitable genes for this essay from previous studies [18, 26]. Target mRNA sequences of IFN- γ, IL-2, GM-CSF and 3 reference genes are amplified using commercially available PrimePCR ddPCR Gene Expression Probe Assays (Biorad) by digital PCR (Biorad).