For STORM imaging, slides were immersed in oxygen-scavenging buffer [glucose oxidase (560 μg/ml), catalase (34 μg/ml), 1% β-mercaptoethanol, 25 mM glucose, 5% glycerol (all from Sigma), and 25 mM Hepes/PBS (Thermo Fisher Scientific) (pH 8); 0.22 μm filter-sterilized], which was refreshed regularly to maintain a low-oxygen environment for the fluorophores. For PALM imaging, slides were kept in PBS. STORM and PALM were performed with an inverted microscope (Nikon Ti Eclipse) fitted with focus stabilization (perfect focus system), a TIRF 100× oil immersion lens [numerical aperture (NA), 1.49], and an electron-multiplying charge-coupled device (EMCCD) camera (Andor iXon Ultra 897). For STORM analysis, images were acquired with a 170-mW 647-nm laser (Agilent Ultra High Power Dual Output Laser, set to 30%) with an 18-ms exposure. The optimal frame number was determined as the point at which reconstructed cluster maps did not change with increasing frame acquisition (for NK cell receptors: up to 30,000 frames). Cells were imaged until the optimal frame number was reached or the cells were bleached. For PALM, images were acquired using a 120-mW 561-nm laser (Agilent Ultra High Power Dual Output Laser, set to 30%) and a 20-mW 405-nm laser (increasing from 0.1 to 10% to maintain the blink rate) with a 50-ms exposure. Frames were acquired until the rate of photoswitching was negligible. STORM analysis of KIR3DL1+ YTS cells was performed separately with an inverted microscope (Leica SR GSD microscope) fitted with a TIRF 160× oil immersion lens (NA, 1.43) and an EMCCD camera (Andor iXon Ultra 897). These images were acquired with a 647-nm laser (set to 30%) with a 15-ms exposure. STED was performed with a continuous-wave STED microscope (Leica TCS SP8 STED CW), a 100× oil immersion lens (NA, 1.40), and a HyD hybrid photon detector (Leica Microsystems). Two-color STED microscopy was achieved by sequential excitation/depletion with wavelengths of 568/660 nm and 495/592 nm (1.5-mW/channel supercontinuum White Light Laser and 1.5-W STED depletion lasers, Leica Microsystems). Time gating of 1.0 to 6.0 ms was applied.

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