Calcein Fluorescence Quenching to Measure Plasma Membrane Water Flux in Live Mammalian Cells

Philip Kitchen, Mootaz M Salman, Mohammed Abir-Awan, Tamim Al-Jubair, Susanna Törnroth-Horsefield, Alex C Conner, Roslyn M Bill

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Aquaporins (AQPs) are membrane channel proteins that facilitate the movement of water down osmotic gradients across biological membranes. This protocol allows measurements of AQP-mediated water transport across the plasma membrane of live mammalian cells. Calcein is a fluorescent dye that is quenched in a concentration-dependent manner. Therefore, on short timescales, its concentration-dependent fluorescence can be used as a probe of cell volume, and therefore a probe of water transport into or out of cells. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Kitchen and Conner (2015). For the underlying methodology development, please refer to Fenton et al. (2010) and Solenov et al. (2004).

Original languageEnglish
Article number100157
JournalSTAR protocols
Issue number3
Early online date2 Nov 2020
Publication statusPublished - 18 Dec 2020

Bibliographical note

©2020 The Author(s). This is an open access article under the CC BY-NC-ND license (

Funding: We acknowledge the UK Biotechnology & Biosciences Research Council (to R.M.B., A.C.C., and P.K. through BB/P025927/1); the Swedish Research Council (to S.T.-H. through 2013-05945); the Crafoord Foundation (to S.T.-H. through 20140811 and 20180916); the Magnus Bergvall Foundation (to S.T.-H. through 2015-01534); M.A.-A. is supported by a studentship co-funded by Aston University and the UK Engineering and Physical Sciences Research Council (EP/R512889/1) to R.M.B.; P.K. is the recipient of an Aston University 50th Anniversary Prize Fellowship: M.M.S. is supported through funding from Biogen to Prof. Tomas Kirchhausen. The graphical abstract was created with


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