Facs canto 1 machine

Expression of the NK cell receptors was evaluated by flow cytometry after exposure to various concentrations of decitabine for 24 h. Mouse anti-human PE-labeled CD56 (clone: HCD56), Percp-labeled CD3 (clone: HIT3a), FITC-labeled NKp46 (clone: 9E2), APC-labeled NKG2D (clone: 1D11), and CD158b (clone: DX27) were purchased from Biolegend (San Diego, CA). Each antibody was added to NK cell cultures and incubated for 30 min at 4°C. Cells were washed twice with phosphate-buffered saline before being subjected to assessment with a FACS Canto I machine (BD science, Franklin Lakes, NJ).

After being exposed to experimentally indicated concentrations of decitabine for 24 h, NK cells cytotoxicity assays were performed as described in detail elsewhere (Gao et al., 2009 (link)). Briefly, NK cells and k562 target cells were mixed at an effector/target ratio of 2:1 (5 × 10⁵: 2.5 × 10⁵) in a total volume of 200 µl in a 96-well plate. After incubation for 2 h, 2 µl of 2 mmol/L monensin (Biolegend) were added and the cells were incubated for an additional 3.5 h. Finally, the cells were collected and stained with anti -CD56, -CD3, and -CD107a (clone: H4A3) antibodies for 30 min at 4°C before being submitted to analysis in a FACS Canto I machine (BD science).