Nk cell isolation kit 2

NK cells were purified from splenocytes prepared from three mice via magnetic beads using a NK Cell Isolation Kit II (Miltenyi Biotec Inc.). The degree of NK cell maturation was determined through flow cytometry by measuring the expression levels of CD3, NK1.1, CD27, and CD11b. To measure NK cell activation, the cell surface expression of CD107a, a marker for NK cell degranulation, and intracellular IFN-γ expression were determined (26 (link)27 (link)). Briefly, NK cells were stimulated with YAC-1 target cells at a ratio of 5:1 or without YAC-1 target cells in the presence of brefeldin A (10 μg/ml) and monensin (6 μg/ml) and incubated at 37°C for 6 h in a CO₂ incubator. To measure the level of NK cell degranulation, FITC-conjugated anti-CD107a Ab was added and the level was measured in CD3⁻NK1.1⁺ cells. In addition, intracellular IFN-γ production was determined in CD3⁻NK1.1⁺ cells via flow cytometry.
To measure NK cell-mediated cytotoxicity, YAC-1 tumor target cell lysis was measured as described previously (28 (link)). Briefly, YAC-1 target cells were labeled with Paul Karl Horan (PKH)-26 and mixed with effector cells at various effector to target ratios. Cells were plated and incubated for 4 h at 37°C in a CO₂ incubator. Following incubation, 7-ADD and FITC-annexin V were added to determine the level of apoptosis induced in target cells via flow cytometry.

Liver‐infiltrating lymphocytes (LILs) were isolated as described.⁽¹⁶⁾ Liver nonparenchymal cells were obtained by digestion in a medium containing 0.1% Liberase (Roche, Switzerland), as described.⁽¹⁷⁾ LSECs were isolated by positive isolation with CD146 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Intrahepatic NK cells were isolated by the NK Cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer’s instructions. The purity of isolated LSECs and NK cells was >90% after isolation.