Mechanical Characterization of Glandular Acini Using a Micro-indentation Instrument.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is responsible for tethering the nucleus to the cytoskeleton, providing a pathway for the cell's nucleus to sense mechanical signals from the environment. Recently, we explored the role of the LINC complex in the development of glandular epithelial acini, such as those found in kidneys, breasts, and other organs. Acini developed with disrupted LINC complexes exhibited a loss of structural integrity, including filling of the lumen structures. As part of our investigation, we performed a mechanical indentation assay of LINC disrupted and undisrupted MDCK II cells using a micro-indentation instrument mounted above a laser-scanning confocal microscope. Through a combination of force measurements acquired from the micro-indentation instrument and contact area measurements taken from fluorescence images, we determined the average contact pressure at which the acini structure ruptured. Here, we provide a detailed description of the design of the micro-indentation instrument, as well as the experimental steps developed to perform these bio-indentation measurements. Furthermore, we discuss the data analysis steps necessary to determine the rupture pressure of the acini structures. While this protocol is focused on the indentation of individual glandular acini, the methods presented here can be adapted to perform a variety of mechanical indentation experiments for both 2D and 3D biological systems.