Mab1850

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.

NK-92 cells were tracked using a fluorescent cell tracker dye (PKH26, Red Fluorescent Cell Linker Kit; Sigma-Aldrich, Saint Louis, MO, USA) according to the manufacturer’s instructions. Dead cells were visualized using propidium iodide (PI) staining (BDA-1000; BIOMAX, Seoul, Korea). For immunofluorescence staining, the cells were first fixed using 4% paraformaldehyde (PFA) or methanol for 20 min and non-specific binding was blocked using 10% normal goat serum for 2 h at room temperature. The samples were then incubated with primary antibodies against α-SMA (1:150, ab5694, Abcam, Cambridge, UK), CD56 (1:150, MA5-11563, Invitrogen), collagen type 1 (1:200, ab6308, Abcam), cytokeratin 19 (1:300, ab52625, Abcam), FAP-α (1:200, ab28244, Abcam), Fibronectin (1:200, ab2413, Abcam), granzyme B (1:100, MA1-80734, Invitrogen), IL-6 (1:200, ab214429, Abcam), NKp46 (1:50, MAB1850, R&D system), perforin (1:200, 14-9994-82, Invitrogen), and TGF-β1 (1:200, ab92486, Abcam) overnight or for 2 days at 4 °C. The samples were washed with phosphate-buffered saline and stained with anti-rabbit or anti-mouse secondary antibodies conjugated with Alexa Fluor 594 (A11012) or 488 (A11008 and A11029). Rhodamine phalloidin (1:500, R415, Invitrogen, Carlsbad, CA, USA) and DAPI (1:1000, D9564, Sigma Aldrich, St. Louis, MO, USA) were used to stain F-actin and nuclei, respectively.

Receptor agonists were adsorbed onto tissue culture plates for 24 hours prior to NK cell harvesting and plating. A total of 300 μL of PBS containing 5 μg/mL anti-NKp46 (R&D Systems; catalog MAB1850), 1.25 μg/mL recombinant MICA Fc-chimera (R&D Systems; catalog 1300-MA), and 1.25 μg/mL recombinant MICB Fc-chimera (R&D Systems; catalog 1599-MB) was added to 24-well plates and incubated at 4°C overnight. For the unstimulated condition, 300 μL of isotype solution (5 μg/mL; IgG2b) in PBS was added in parallel. 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. Then 1 mL (1 × 10⁶ NK cells) was added to each well of the previously prepared 24-well plates. After 3 or 7 days, NK cells were harvested, washed with PBS, counted, and used for various experiments. For exhaustion assays spanning 7 days, NK cells were harvested, washed, and replated onto identical plates on day 3.