Assessment of Calcein Dye Method to Observe Cell Volume Regulation

Cell volume regulation (CVR) is a crucial mechanism for the maintenance of homeostasis of cells under significant osmotic challenges. When cells are swollen or shrunken in a non-isotonic medium they initiate a regulatory mechanism to restore to their initial volume. Swollen cells in a hypotonic solution show decreases in cell volume by releasing osmolytes and water, through a regulatory volume decrease (RVD) mechanism. On the contrary, shrunken cells in a hypertonic solution exhibit a regulatory volume increase (RVI) by up-taking osmolytes and water. The whole process of CVR is complicated and involves diverse cell sensory mechanisms of a volume change and complex signal pathways that eventually leads to the activation of ion channels or co-transporters. For a better understanding of CVR, it is therefore necessary to develop a method that can effectively trace changes in cell volume using a large number of cells. Calcein dye method has been used to trace cell volume change but contradictory results have been observed and reported. To understand the unusual behavior of calcein fluorescence, the research has been focused to validate the use of calcein dye in measuring cell volume change in non-isotonic solution. In addition, it was demonstrated that the volume changes of single cells could be alternatively monitored by tracing the changes in the areas of single cells obtained from time-lapse fluorescence micrographs