期刊
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
卷 298, 期 1, 页码 F224-F230出版社
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajprenal.00463.2009
关键词
aquaporin-2; calcein; Madin-Darby canine kidney cells
资金
- Lundbeck Foundation
- Nordic Centre of Excellence
- Danish Medical Research Council
- Novo Nordisk Foundation
- Carlsberg Foundation
- Marie Curie Intra-European Fellowship
- Danish National Research Foundation (Danmarks Grundforskningsfond)
- Lundbeck Foundation [R54-2010-5398] Funding Source: researchfish
Fenton RA, Moeller HB, Nielsen S, de Groot BL, Rutzler M. A plate reader-based method for cell water permeability measurement. Am J Physiol Renal Physiol 298: F224-F230, 2010. First published November 4, 2009; doi: 10.1152/ajprenal.00463.2009.-Cell volume and water permeability measurements in cultured mammalian cells are typically conducted under a light microscope. Many of the employed approaches are time consuming and not applicable to a study of confluent epithelial cell monolayers. We present here an adaptation of a calcein-quenching-based approach for a plate reader. A standard curve of fluorescence intensities at equilibrium has been recorded, following a shift from 285 mosmol/kgH(2)O to a series of altered extracellular osmolyte concentrations, ranging from final concentrations of 185 to 585 mosmol/kgH(2)O, by changing buffer D-mannitol concentrations. Similarly, according average cell volumes have been measured in suspension in a Coulter counter (particle-sizing device). Based on these measurements, we have derived an equation that facilitates the modeling of cell volume changes based on fluorescence intensity changes. We have utilized the method to study the role of a carboxyl-terminus aquaporin (AQP)-2 phosphorylation site, which is known to affect AQP2 membrane trafficking, in heterologous type I Madin-Darby canine kidney cells. We find that water permeability in cells expressing phosphorylation site mutants was in the following order: AQP2-S256D > AQP2 wild-type > AQP2-S256A. We propose that the method can be applied to study AQP function and more generally to study cell volume changes in adherent cell lines. Furthermore, it should be adaptable for AQP inhibitor screening in chemical compound libraries.
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