NK Cell Isolation, cultivation and cytotoxicity assay
NK cells were isolated from MNCs of patients with AML before the initiation of induction chemotherapy (n = 9) by selecting CD56+ cells using magnetic bead selection (Miltenyi Biotech, USA). The purity of the isolated cells was calculated and confirmed by flow cytometry and specific antibody against CD56+. Then, NK cells were divided into five groups (NK cells without any factor, NK cells that received Human IL-15, NK cells that received IL-15 and PD-1 blocker, NK cells received IL-15 and Hsp70, and the last group of NK cells received IL-15, PD-1 blocker and Hsp70). These NK cell groups from patients with AML were assessed for their capacity to kill NK cell-sensitive KG1 cells. The KG-1 cell line was inactivated using Mitomycin C (20 μg/ml; 2 × 106cells per well) to inhibit their proliferation, then they were labeled with Calcein AM for 45 minutes of incubation at 37 °C in 5% CO2. The labeled cells were washed with PBS-, and resuspended in X-X IVO medium with 10% FBS. KG1 cells were co-cultured at an effector: target (E: T) ratio of 10:1, which incubated for 24 hours at 37 °C in 5% CO2. Cytotoxicity level was evaluated with calcein/propidium iodide (PI) staining by flow cytometry technique. The control groups consisted of tumor cells grown in the same media without NK cells exposure.