Measuring Dynamic Cell Volume In Situ by Confocal Microscopy

Regulation of cell volume is a fundamental homeostatic mechanism in the face of osmotic stress (1 ,2 ). One approach to understanding aspects of cell volume regulation involves the removal of cells from the matrix and manipulation in culture (3 ). Measurements are often also needed from cells within intact tissue that are operating in their correct physiological context. These include the effects of cell-cell interactions and the mechanical, ionic, and physiological effects of the extracellular matrix (ECM). The procedure described here is an in situ approach to volume measurement using an organ culture system, which maintains tissue integrity and hence the spatial organisation of cells in the ECM (4 ). We present a comprehensive protocol for investigating volume regulatory behaviour using confocal laser scanning microscopy (CLSM). Although we focus on articular and fetal growth plate cartilage tissues, the protocols can be applied to other intact animal, plant, and fungal tissues (5 ). As we show below, many factors must be taken into account in the choice of experimental parameters, there are no “best settings” that work for all tissues, in all cases.