Investigating Integrin Regulation and Signaling Events in Three-Dimensional Systems

There has been much recent interest in working with cells cultured in three-dimensional (3D) matrices to better model the properties of the extracellular matrix environment found in vivo. However, working within 3D matrices adds several difficulties to experiments that have become routine in two-dimensional (2D) culture systems. Biochemical approaches are made difficult by the presence of milligram quantities of matrix protein, while cell biology approaches are more difficult to assess and image. Moreover, 3D imaging adds complexity to fluorescence studies, including the inherent challenge of a 3D volume as opposed to a 2D image, increased depths of field, and problems of light scatter. The purpose of this chapter is to provide a few overall strategies for working within 3D culture systems, focusing on biochemical and molecular imaging approaches.