Experimental approaches for studying cell water and volume regulation in apoptotic cells

Shrinkage and water loss are unique features of apoptosis. Despites decades of research, their exact roles, as well as the mechanisms of development, are poorly understood. Research in this area requires the ability to measure cell volume, intracellular water content, intracellular ion concentrations and the assessment of specific membrane permeability. Cell volume The volumes of adherent cells can be conveniently measured by transmission‐through‐dye (TTD) microscopy. This method is based on exclusion of a strongly absorbing extracellular dye by intact cells. The method can be realized on any transmission light microscope at nearly zero cost. TTD provides accurate measurements, even on severely deformed cells (which are common during apoptosis) and is compatible with fluorescence. Cell water Cell water is another essential parameter involved in apoptosis. Apoptotic shrinkage can occur by water expulsion as well as by cell fragmentation, and to discriminate between these two mechanisms, direct water measurements must complement measurements of the cell volume. Intracellular water is directly related to the average refractive index; the latter can be obtained from the the volume (which can be determined by the TTD) and from the delay of the light wave crossing the sample. A simple and effective way to measure the phase is based on the transport‐of‐intensity equation (TIE). Two transmission brightfield images are collected at different distances to the sample, and the phase is restored by solving the TIE. A Matlab‐based code for solving TIE is available. Ion measurements Monovalent ions play a major role in volume regulation. Because the properties of fluorescent ion probes depend on the environment, they have to be calibrated in situ . Ionophores are often used to control intracellular milieu; however, equalization of intracellular and extracellular ion concentrations would require the destruction of the membrane potential and a harsh treatment of cells. One way to avoid it is to measure the actual membrane potential y simultaneously with the ion of interest. The process can be presented by the following flow‐chart: Membrane permeability Apotosis can alter membrane permeability not only for inorganic ions, but for certain organic substances, such as choline. Some of the common cellular stains utilize same pathways and can be used to assess the cell permeability. More work is needed to characterize the channels involved in these processes. Support or Funding Information The work was partially funded by KSU Research Council grant