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.