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MATERIALS AND METHODS Materials RPMI-1640 medium, penicillin, streptomycin, phytoheamagglutinin (PHA), and trypan blue (TB) were from sigma (USA). Fetal calf serum (FCS) was from Gibco (USA). Interleukin-2 (IL-2) and Interferon- �� (IFN-��) standard ELISA kit was obtained from R & D company (USA). Propranolol was a kind gift from HAKIM Pvt. Co. Ltd (Tehran, Iran). Microtiter plates, flasks and tubes were from Nunc (Falcon, USA). Cell lines Human leukemic T cells [Molt-4 (NCBI C149) and Jurkat (NCBI C121)], were obtained from NCBI (National Cell Bank of Iran, Pasteur Inst. of Iran, Tehran). The cells were maintained in RPMI-1640 medium supplemented with 10% FCS in 5% CO2 at 37��C. Preparation of propranolol Propranolol was dissolved in RPMI-1640 medium and stored at -20��C until use. Drug was diluted in culture medium to prepare the needed concentrations before use. Cell culture and treatment The method has been Selleckchem SCR7 described in detail elsewhere.15 Briefly, the human leukemic cells were cultured in RPMI-1640 medium supplemented with 10% FCS, penicillin (100 IU/ml) and streptomycin (100 ?g/ml) at 37��C in 5% CO2. The cells were seeded at a density of 2��106 cell/ml and then incubated with different concentrations Entinostat molecular weight of propranolol (0.03- 30 ?M) in the presence or absence of PHA (10 ?g/ml) for 48 hours. The supernatants of cell culture media were collected and used for IL-2 and IFN-�� assay. All experiments were done in triplicate. Cytokines assay The amount of IL-2 and IFN-�� secreted in the cell culture supernatants by human leukemic T cell lines (Molt-4 and Jurkat) was measured with the Quantikine human enzyme-linked immunosorbent assay (ELISA) kits (R & D systems) according to the manufacturer��s instructions. This assay uses the quantitative sandwich enzyme immunoassay technique. Complete RPMI medium was used as control and human recombinant IL-2, and IFN-�� were Mianserin HCl employed as standard for drawing the standard curves. Statistical analysis Effect of the drug on each cell line was performed in three independent experiments and the results were expressed as mean �� SEM. Statistical comparisons among groups were made by analysis of variance (ANOVA). P