Analysis of cellular morphology by confocal microscopy and a membrane-intercalating fluorescent dye PKH67

Abstract

Cells of the body are exposed to anisosmotic environments under various physiological and pathological conditions, which cause mobilization of water and changes in cellular volume and morphology; these changes provide an indication of the cellular environment changes and predict the subsequent cellular damage. We have constructed confocal microscopy-based methods to analyze osmotic stimuli-induced changes in cell dimension and morphology using a membrane-bound fluorescent probe, PKH67. Low osmolarity stimulation of the renal tube derived MDCK cells resulted in an initial increase in their cell volume, which was followed by a subsequent decrease towards initial value. Transient exposure to high osmolarity formamide solution of mouse ventricular cardiomyocytes resulted in complete loss of their transverse tubules (T-tubules). Confocal microscopy in combination with a membrane-intercalating dye was shown to be simple, accurate and free of cellular damage, and would be useful for further studies on the regulation of cellular volume and morphology.