NK cell activity assay by LDH

The human erythroleukemia K-562 cell line (Chinese Academy of Sciences, Shanghai, China) was used as the control cells in this assay. Briefly, K-562 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) and then plated at a concentration of 1×105 cells/ml in U-bottom 96-well plates (Corning, Inc., Shanghai, China). Murine spleens were harvested and extracted by centrifugation at 1,000 × g for 10 min at 20±2°C, and splenocytes were plated at 1×10⁵ cells/ml for an effector to target ratio of 50:1 on day 2 post radiation. The cytotoxic activity of NK cells in each of these groups used from the mice splenocytes was measured at the same day using a standard in vitro lactate dehydrogenase (LDH) release assay as previously described (13). The plates were incubated for 20 h at 37°C and 5% CO₂, and then centrifuged for 5 min at 1,000 × g at 20±2°CC. Subsequently, 100 µl supernatant was moved to a new plate, followed by addition of 100 µl LDH kit (P1240; R&D Systems, Inc.) and 30 µl HCl (1 M) to each well. Finally, the optical density (OD) was measured using an Infinite M200 PRO automatic microplate reader (Tecan Group Ltd., Männedorf, Switzerland) at a wavelength of 490 nm. The percentage of cytotoxicity was calculated using the following formula: Cytotoxicity (%)=(sample OD-spontaneous OD)/(maximum OD-spontaneous OD) ×100 (13).