Table 2.

Various assay methods used in the measurement of AQP function and the validation of AQP inhibitors. Their benefits and limitations are shown.

Assay Type	Assay Principle	Benefits	Limitations	References
Epithelial	Epithelial cells are placed on supports, solutes are added and transepithelial flux is measured by the height of the fluid in capillary tubes	Assists AQP identification and characterization in tissues Individual membrane permeability is calculated	Not very robust Measures paracellular as well as cellular water flow	[94,95]
Osmotic Swelling	Osmotic gradients are used to cause flux of solutes and water in cells endogenously-expressing or transfected with AQP genes; cell volume changes are measured	Well-established technique Oocytes have low intrinsic water permeability Other cells can be used Inward and outward gradients possible	Preparation techniques vary causing discrepancies in results between laboratories Not very reproducible Variability between oocytes	[94,96,98]
Microscopy	Most often fluorescent dyes are used that permeate the plasma membrane and are cleaved by esterases. As osmotic shock occurs, the fluorescence is altered, which is measured and used to calculate cell volume changes	Linear relationship produced between fluorophore and cell volume changes More accurate than counterparts Very sensitive Robust	Thickness of cell-line monolayer can produce variability in results Fluorophore may not always stay within the cell depending on cell-line	[94,99,104]
Stopped-Flow Spectroscopy	Suspensions of cells or vesicles are mixed with various osmotic solutions and flux of water causes volume changes, which result in altered light scattering. The linear relationship between optical properties and cell volume allows permeability to be calculated; alternatively fluorophores can be used.	Fast kinetics measured Can use cells, vesicles and proteoliposomes Linear relationship between optical properties and volume changes	Variability in dead time and mixing ability can cause problems with reproducibility	[94,99,101]
Computational Methods	High resolution atomic structures of AQPs are used and molecular dynamics simulations allow in silico measurement of water permeability	Provides new insights into AQP structure and function	High-quality structures not always available May disagree with experimental data	[94,103]