| Method |
Through the use of particle nanotracking, researchers detect and quantify the micro-organization as well as the viscoelastic properties of the intranuclear region in single, live, interphase somatic cells.Glass coverslips were cleaned and treated with 0.1-% poly-L-lysine (Sigma, St Louis, MO), and then coated with 20 µg/ml fibronectin (Calbiochem, San Diego, CA). Cells were seeded on those coverslips and allowed to grow for 24-48 hours before experimentation. Seeded coverslips were placed in a parallel-plate flow chamber (Glycotech, Rockville, MD), which imposed calibrated shear flows, typically for 25-35 minutes, as indicated. The flow chamber was mounted on an inverted microscope (Nikon) and enclosed in an environmental chamber maintained at 37°C and 5% CO2. Cells were verified to remain viable after application of the shear. The time-dependent positions of the nucleoli centroids were obtained using the particle-nanotracking system described above. The cell centroid was determined by tracing the edges of the cell, visualized by phase-contrast microscopy. The set of distances between nucleoli was used as a marker of structural coherence of the nucleus (Paddock and Albrecht-Buehler, 1986a Paddock and Albrecht-Buehler, 1986b) the distance between cell centroid and nucleus centroid was used as a marker for the relative stiffness of cytoplasm and nucleus. |