3D Collagen Cytotoxicity Assay for NK Cells

Target cells (K562-pCasper cells) were embedded into 2 mg/mL of pre-chilled neutralized bovine type I collagen solution (Advanced Biomatrix) in a 96-well plate. The collagen was solidified at 37 °C with 5% CO₂ for 40 min, after which NK cells were added to the top of the collagen as effector cells. The cells were visualized using ImageXpress Micro XLS Widefield High-Content Analysis System (Molecular Devices) at 37 °C with 5% CO₂. For the 3D killing assay, as described previously [47 (link)], killing events were visualized every 20 min for 36 h, and live target cell numbers were normalized to hour 0 based on area. For live cell imaging to determine time required for killing and the average kills per NK cell, the cells were visualized every 70 s for 14 h and tracked manually. ImageJ software was used to process and analyze the images.