Sildenafil Modulates CD56+ Cell Culture

Isolated CD56-positive cells were cultured in 24-well plates (SPL Life Sciences Co., Ltd., Korea), in Opti-MEM Reduced Serum Media (Gibco, Life Technologist) containing 10% FCS (Sigma-Aldrich), 1 U/ml penicillin/streptomycin/100 μg/ml (PAA), 2 mM glutamine (Sigma-Aldrich), 1 mM sodium pyruvate (Fluka), nonessential amino acids (Gibco, Thermo Fisher Scientific), 55 nM 2-mercapthoethanol, 10 ng/ml recombinant human IL-15 (Sigma-Aldrich), 2 ng/ml recombinant human TGF-β-1 (R&D), and 1 μM 5-aza-2′-deoxycytidine (Sigma-Aldrich) in hypoxic (94% N₂, 5% CO₂, and 1% O₂) environment [9 (link)]. The cells were cultured in 1 × 10⁶/ml concentration in two variants: with and without 400 ng/ml of sildenafil citrate (Sigma-Aldrich). The concentration of sildenafil used in the experiments equaled the blood concentration of sildenafil after the oral administration of 200 mg of Viagra in healthy men [35 (link)]. After 7 days of culturing, the cells were harvested for flow cytometry analysis.