In vitro NK Cell Migration Assay

For in vitro migration assay a transwell system (Costar; Corning, USA) was used. Here, the NK cell attracting chemokine CXCL12 (250 ng/ml, R&D Systems) was added to the lower chamber in a total volume of 600 μl chemotaxis medium (RPMI containing 25 mM HEPES and 0.5% BSA). Total splenocytes obtained from untreated and Lm infected mice, respectively, were loaded to the upper chamber at a density of 5 × 10⁵ cells in 100 μl chemotaxis medium. Cells were allowed to migrate across the pores of the transwell inserts (pore size 5 μm) at 37°C and 5% CO₂. After 4 h, transmigrated cells from a pool of three wells were harvested by centrifugation (5 min, 300 ×g), resuspended in 100 μl PBS containing 2 mM EDTA, stained with trypan blue and counted using a hemocytometer. The percentage of NK cells before and after transmigration was analyzed by flow cytometry. Briefly, cells were first incubated with anti-CD16/CD32 (2.4G2, BD Pharmingen) for 10 min on ice followed by incubation with anti-CD3ε (FITC, 145-2C11, BD Pharmingen), anti-NK1.1 (APC-Cy7, PK136, BioLegend), anti-NKp46 (BV450, 29A1.4, BD Bioscience) for 30 min on ice. After washing, analysis was performed on a BD LSRFortessa (BD Biosciences) flow cytometer. Data are expressed as percentage of input calculated by the following equation: migrated NK cell number/input NK cell number.