Morphological and biochemical analysis of Rac1 in three-dimensional epithelial cell cultures.

Abstract Rho GTPases are critical regulators of epithelial morphogenesis. A powerful means to investigate their function is three‐dimensional (3D) cell culture, which mimics the architecture of epithelia in vivo . However, the nature of 3D culture requires specialized techniques for morphological and biochemical analyses. Here, we describe protocols for 3D culture studies with Madin‐Darby Canine Kidney (MDCK) epithelial cells: establishing cultures, immunostaining, and expressing, detecting, and assaying Rho proteins. These protocols enable the regulation of epithelial morphogenesis to be explored at a detailed molecular level.