Ldh detection kit

Natural killer cells were freshly isolated from whole EDTA blood using the MACSxpress NK isolation kit following the manufacturer instruction. Monoclonal antibodies were serially diluted tenfold in AIM-V medium from 1 μg ml⁻¹ to 0.001 μg ml⁻¹. Target cells (A549-H1HA, A/California/04/2009) were added in a round-bottom 384-well plate at 7.5 × 10³ cells per well in 23 μl, then serially diluted antibodies were added to each well (23 μl per well), and the antibody–cell mixture was incubated for 10 min at room temperature. After incubation, human natural killer cells were added at a cell density of 4.5 × 10⁴ per well in 23 μl (effector to target ratio of 6:1). Control wells were also included that were used to measure maximal lysis (containing target cells with 23 μl of 3% Triton X-100) and spontaneous lysis (containing target cells and effector cells without antibody). Plates were incubated for 4 h at 37 °C with 5% CO₂. Cell death was determined by measuring lactate dehydrogenase (LDH) release using an LDH detection kit (Roche) according to the manufacturer’s instructions. Using a kinetic protocol, the absorbance at 490 nm and 650 nm was measured once every 2 min for 8 min. The percentage of specific lysis was determined by applying the following formula: (specific release − spontaneous release)/(maximum release − spontaneous release) × 100.