Flow cytometery

Blood samples were collected from refractory/relapsed
patients, responsive patients and healthy donors. PBMCs
were separated on a Ficoll-Hypaque gradient. NK cells
were purified by negative selection, using NK cell
isolation kit and LS columns (MiltenyiBiotec, Germany).
The purity of N cells was confirmed as 85-90% by
flow-cytometery (BD Company, USA) using PE-cy5labeled
anti-CD56 and FITCI-labeled anti-CD3 (both
from eBioscience, USA). The range of CD3 positive
cell contamination in purified NK cells was 10-15%.
To obtain polyclonal NK cell populations, PBMCs were.-ray irradiated (25 Gy) and they were used as autologousfeeder cells for co-culture with NK cells at a ratio of 4:1
feeder-NK cell. NK cells were expanded in Cellgro SCGMserum-free media (CellGenix, USA) supplemented with 5%
human AB serum, 10% fetal bovine serum (FBS, Gibco,
USA), 50 U/ml penicillin, 50 µg/ml streptomycin, 500 IU/
ml recombinant human interleukin-2 (IL-2, MiltenyiBiotecAG, Germany), 10 ng/ml recombinant human interleukin-15(IL-15, MiltenyiBiotec AG, Germany) at a density of 5×10⁵
cells/ml in T-25 flask for 3 weeks.

The intracellular ROS levels were determined by using a ROS Assay Kit (Beyotime, Shanghai, China) following the protocols and the methods described previously [29 (link)]. Target cells were treated under different conditions and incubated with 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) for thirty minutes at 37 °C. Then cells were harvested for flow cytometery (BD, USA) analysis [30 (link),31 (link)].