Mab004

Receptor agonists were adsorbed onto tissue culture plates for 24 hours prior to NK cell harvesting and plating. A total of 300 μL of the following PBS solutions were added to 24-well plates and incubated at 4°C overnight: anti-NKp46 (5 μg/mL; [R&D Systems; catalog MAB1850]), recombinant MICA Fc-chimera (1.25 μg/mL [R&D Systems; catalog 1300-MA]), recombinant MICB Fc-chimera (1.25 μg/mL [R&D Systems; catalog 1599-MB]), and isotype (20 μg/mL IgG2b; [R&D Systems catalog MAB004]); anti-NKp46 (5 μg/mL; [R&D Systems catalog MAB1850]), recombinant MICA Fc-chimera (1.25 μg/mL [R&D Systems; catalog 1300-MA]), recombinant MICB Fc-chimera (1.25 μg/mL [R&D Systems; catalog 1599-MB]), and anti-NKG2A (20 μg/mL; [Beckman Coulter catalog IM2750]); or isotype only (25 μg/mL IgG2b [R&D Systems catalog MAB004]). After 24 hours, plates were washed twice with PBS and NK cells were added; NK cells were counted and resuspended in B0 medium supplemented with 1 ng/mL IL-15 (R&D Systems) at a density of 1 × 10⁶ cells/mL. NK cells were added (1 × 10⁶ cells/well) to each well of the previously prepared 24-well plates. After 7 days (including replating at day 3 as described previously) NK cells were harvested, washed with PBS, counted, and used for various experiments.

A549 cells were seeded onto 6-well plates and transfected with siRPS16 (or siNC control). After 48h of transfection, the cell was infected with influenza A/WSN/1933 (MOI = 0.1), the cells were maintained with medium containing IFN-β neutralizing antibody (2 µg/ml) (R&D system, MAB8141) or same concentration of antibody iso-type control IgG (R&D system, MAB004). The NP protein expression was tested by western blot assay at 24 h post infection.

Migration assays were carried out in a 24-well transwell system equipped with porous (8 μm) polycarbonate membranes. 2 × 10⁵ MB cells were resuspended in 600 μL of growth medium supplemented with 1% FBS and plated into the lower chamber of the transwell system; 5.0 × 10⁴ BMSC cells were resuspended in 200 mkl of growth medium supplemented with 1% FBS and plated into Transwell inserts which were then placed into another transwell system with the lower chamber filled with 600 μl of serum-free medium. After 24 hours the cells on the inserts or in lower chambers were transfected as indicated, 6 hours after the transfection the media was changed and the inserts with BMSC, were placed into the wells with MB cells and incubated at 37 °C for 24 h. When indicated, CXCR4 (Abcam, #ab10403) or SDF1 (CXCL12) (Abcam, #ab9797) antibodies were added to the medium at 10 mkg/ml and 4 mkg/ml concentrations respectively. The inserts were then discarded, and upper sides of the filters were swabbed to remove the cells that did not cross the membrane. The cells present on the lower side of the filters were then fixed in 4 % paraformaldehyde, stained with DAPI and the counted under the microscope. All the experiments were performed in duplicate. The following control antibodies were used: rabbit IgG control (AB-105-C, R&D systems) and mouse IgG2b isotype control (MAB004, R&D systems).