Real-Time Imaging of Anti-CD276 mAb Binding

We investigated the surface binding and internalization of our anti-CD276 mAb using live-cell confocal imaging following established protocols in our previous publications [29 (link),30 (link),33 (link)]. H460-Fluc and human NSCLC were cultured in 35 mm glass bottom dishes (Cellvis, Mountain View, CA, USA) at a density of 1 × 10⁴ cells per dish in 1.5 mL of medium. To visualize the cytoplasm and nucleus, BacMam GFP Transduction Control (Invitrogen) and NucBlue™ Live ReadyProbes™ Reagent (Invitrogen), respectively, were used for staining following manufacturing protocols. Next, the AF647-labeled CD276 mAb was added to the cells at a final concentration of 1 or 5 µg per mL. Live-cell images were captured at 0–24 h after the addition of CD276 mAb-AF647 using a Nikon A1R-HD25 confocal microscope (Nikon USA, Melville, NY, USA). This experimental approach allowed us to dynamically monitor the binding and internalization of CD276 mAb into NSCLC over different time points.