NK Cell-Mediated Cytotoxicity Assay

NK cytotoxic activity was conducted as previously described [52 (link)]. NK cells were labelled with Paul Karl Horan (PKH-26) (3.5 μl/test) (2 × 10^− 6 M) for 5 min (Sigma-Aldrich, St. Louis. MO, USA) and incubated with K562 cells for 4 h at 37 °C with 5% CO₂ in RPMI-1640 supplemented with 10% FBS. The concentration of NK cells and K562 cells were adjusted to 5 × 10⁵ cells/ml and 1 × 10⁶ cells/ml, respectively. During incubation, cells were combined at E:T ratios of 12.5:1 and 6.25:1 in addition to controlling samples. Control and RTX treated cells were stained using Annexin V (2.5 μl/test) (BD Bioscience, San Jose, CA, USA) and 7-amino-actinomycin (7-AAD) (2.5 μl/test) (BD Bioscience, San Jose, CA, USA) to determine apoptosis using flow cytometry analysis recording 10,000 events (Additional file 1). E:T ratio of 12.5: 1, 6.25 and control were used to assess cytotoxic activity [38 (link), 40 , 53 (link)]. NK cytotoxic activity was calculated as percent specific death of the K562 cells for the two E:T ratios as previously described [40 , 52 (link)]. The percentage of target cell lysis was calculated from: $$\mathit{\text{Cytotoxicity}}\left(\%\right)=\frac{\left(\mathit{\text{early stage apoptosis}}+\mathit{\text{late stage apoptosis}}+\mathit{\text{dead}}K562\mathit{\text{cells}}\right)}{\mathit{All}K562\mathit{\text{Cells}}}\times 100$$