Af488 labeled phalloidin

Isolated human monocytes were labeled with allophycocyanin-conjugated mouse monoclonal anti-CD14 and PE-conjugated mouse monoclonal anti-CD16 or isotype controls (all from eBioscience, Hatfield, U.K.), according to manufacturer’s protocols. Murine blood, peritoneal lavage, or air-pouch lavage cells were labeled with PE-conjugated rat monoclonal anti-Ly6G or anti-Gr1, PE-Cy5–conjugated rat monoclonal anti-CD115, and allophycocyanin-conjugated rat monoclonal anti-F4/80, or isotype controls (all from eBioscience), all according to manufacturer’s protocols. In all cases, 20,000 events were acquired using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo analysis software (Version 9.6.3; Tree Star, Stanford, CA). The human and murine gating strategies for cellular analysis are shown in Supplemental Fig. 2A and 2B, respectively. Actin polymerization was assessed through binding of AF488-labeled phalloidin (Invitrogen), according to manufacturer’s protocols.

NK cell–cancer cell conjugates were formed by incubating NK cells with cancer cells for 10, 20, or 40 min before fixation with 4% PFA/PBS for 20 min at RT. Conjugates were permeabilized with 0.1% Triton X-100 (Sigma) for 10 min at RT, blocked with 3% BSA in PBS for 1 h at RT, and stained with anti-pericentrin antibody (1 µg/mL; ab4448; Abcam) in blocking buffer for 2 h at 4 °C. Conjugates were then washed with PBS, stained for 1 h at RT with AF568-conjugated goat anti-rabbit secondary antibody (5 µg/mL; Invitrogen), AF647-conjugated anti-perforin antibody (0.625 μg/mL; dG9; Biolegend), AF488-labeled phalloidin (10 µg/mL; Invitrogen), and BV421-conjugated anti-CD56 antibody (1.5 μg/mL; HCD56; Biolegend), washed with PBS, and transferred to a PLL-coated eight-chambered glass coverslip. Imaging was performed by confocal microscopy (SP8, Leica Biosystems) using a 63×/1.40 NA oil immersion objective with excitation using 405-, 488-, 568-, and 647-nm laser lines. Images were analyzed using ImageJ (58 (link)). The distance of the immune synapse to the MTOC and back of the cell was measured manually. The length of the immune synapse was quantified by measuring the area of contact between an NK cell and target cell based on actin staining at the z-plane in which contact was greatest.