FCF blocks impairs fibronectin-induced cell spreading and abrogates conjugate formation. (A) Primary NK cells were treated with diluent (DMSO) or 100 mM FCF for 2 h and then dropped onto fibronectin-coated coverslips and allowed to adhere and spread for 15 min at 37°C in a humidified chamber. The coverslips were subsequently fixed and stained with anti–septin 1 (red) and phalloidin to detect F-actin (green). Representative images from three independent experiments are shown. Scale bar, 10 µm. (B) The diameter of the cell along its longest access was measured using ImageJ. Data represent three independent experiments in which the diameter of 50 cells was calculated. ***, P < 0.05 as determined using the unpaired two-tailed Student’s t test. (C) NKL cells were stained with Calcein-AM and then allowed to form conjugates at 37°C with CMAC-labeled 721.221 cells over the specified times and then fixed. The formation of conjugates (CMAC + Calcein-AM⁺) with respect to the total number of NKL cells (Calcein-AM⁺) was assessed using two-color FC. Results shown are the representative of three independent experiments, each performed in triplicate.