Immunological synapse observation by immunofluorescence microscopy

HEK cells transfected with hCD48 or the corresponding empty vector were first labelled with CellTracker Blue CMAC. After washing, labelled HEK cells were mixed for 10 min at 37°C and a final volume of 500 μL with 1x10⁵ YT cells at a ratio of 1:1 to allow conjugate formation. The suspension was placed on coverslips coated with poly-L-lysine (Sigma-Aldrich) for 10 min at 37°C, washed with PBS, fixed with 4% formaldehyde for 10 min, and permeabilized with 0.05% Triton X-100 for another 10 min. Subsequently, samples were blocked with 20% rabbit serum and 6% fetal bovine serum in PBS and incubated with Alexa Fluor 488 Phalloidin (Invitrogen) and the primary anti-perforin mAb, followed by a secondary antibody goat anti-mouse IgG (H+L)-Alexa Fluor 555. Coverslips were mounted in ProLong Gold antifade reagent (Invitrogen). Fluorescence images were acquired using a Nikon Optiphot-2 microscope (Nikon Corp.). Synapse stages were defined as: 0, conjugates lacking actin polymerization and perforin polarization; 1, conjugates with polymerized actin, but missing perforin polarization; 2, conjugates with actin polymerization and perforin clustered at the immune synapse. When indicated, NK cells were pre-incubated with 10 μg/mL of A43-Fc or CTL-Fc fusion proteins, at 37°C during 30 min prior to being mixed with the target cells.