In vitro chemotaxis assay

Neuro probe blind well chemotaxis chambers with a lower well volume of 200 µL were used. In the lower wells of the chamber, 200 µL of medium were placed in the presence or absence of chemokines, HCT-116 supernatants, or K562 supernatants. In the antibody neutralization experiments, antibodies specific for CCL27, CCL28, or CXCL16 were incubated with the supernatants for 1 h. Upper and lower compartments were separated by 8 µm Nuclepore polycarbonate filters (Whatman International Ltd., Maidstone, UK), and 2 × 10⁵ NK92 cells were placed in the upper compartments, whereas the supernatants either untreated or pretreated with the neutralizing antibodies were placed in the lower compartment of the chambers. After the 2 h incubation period, filters were removed and dehydrated with methanol, after which they were stained with 15% modified Giemsa stain for 25 min and fixed on glass slides. NK92 cells were counted in × 10 power field, and an average count was calculated for each sample. Migration index (MI) was calculated using the equation where the number of cells migrating towards chemoattractant (chemokines or supernatant) is divided by the number of cells migrating towards medium only. Mean ± SEM of 3–4 experiments was calculated for each experiment, where 4–6 filters were used and averaged in each experiment.