Videomicroscopy of dNK-JEG-3 interactions

JEG-3 were cultured in Delta T dishes (Bioptechs) coated with fibronectin (20 μg/ml), which were adapted to an Olympus FV1000 4-channel Confocal Imaging system. dNK were pre-labeled with Vybrant DiO at 1:1000 for 30 min in serum-free RPMI and then washed 3 times. After temperature calibration (37°C), the JEG-3 Delta dish was placed in the stage, and dNK were added at T=0. Cells were imaged with the 60x objective with transmitted light and 488 nm laser with emission filter of 500–545 nm, and images were taken every minute for up to 20 minutes. Images were processed using FV10-ASW software version 03.00.01.15.