Membrane-labeled MDCK cells and confocal microscopy for the analyses of cellular volume and morphology.

A clone of Madin-Darby canine kidney (MDCK) cells whose cell membrane was stably labeled with expressed cyan fluorescent protein (CFP) was established, and changes in their volume and shape induced by hyposmotic stress were analyzed with confocal microscopy. The membrane-targeted CFP was present not only on the cell membrane but also in the endoplasmic reticulum and Golgi apparatus, but was excluded from the mitochondria and cell nucleus. During hyposmosis, the initial swelling and the following regulatory volume decrease could be accurately measured by summation of the cellular volume in every confocal slice crossing the cell at different heights. Changes in the cellular height roughly paralleled the changes in cellular volume when the mean value was compared, but dissociation as much as 30% was observed for individual cells due to changes in cell shape. The present experimental system, which enables direct measurement of cell volume and simultaneous observation of various intracellular phenomena, would be useful for further investigation of cellular volume regulation.