Glass bottom dishes 1.5

Chemotactic sensing assays were performed as previously described (Mouneimne et al., 2006 (link)). Briefly, cells were plated on glass-bottom dishes (1.5; MatTek) coated with 10 µg/ml fibronectin (Corning) and allowed to adhere for ∼24 h. Before imaging, imaging buffer was added to cells (full medium supplemented with 10 mM Hepes). A micromanipulator (InjectMan NI2; Eppendorf) was fitted with a glass micropipette (Femtotip; Eppendorf), which was loaded with 10 nM IGF with 3-kD fluorescently labeled Dextran clarified by 0.2-µm filters. A chemotactic gradient was generated using a pressure-regulated microinjection system at 25pi with continuous flow (FemtoJet; Eppendorf). Micropipettes were placed ∼25 µm away from nonleading edges of single cells, and IGF was released from the micropipette for 2 min. Experiments were imaged on a Ti-E inverted microscope, with a 60× Plan Apo 1.40 NA objective, and an ORCA-Flash 4.0 V2 CMOS camera, a motorized stage, and an environmental chamber. Imaging was done at 37°C with ambient 5% CO₂. Stimulated cells were imaged by DIC microscopy at a 1-s frame rate for 30 min. Wide-field fluorescence, captured at a 1-min frame rate, was used to visualize the chemotactic gradient. Chemotactic sensing was quantified using the sensing index and turning angles of manual traces done in ImageJ (see Sensing index and turning angles).