Abstract
Calcein is an intracellular fluorescent probe that has been used as an indicator of cell volume in several previous studies. These studies have reported two different fluorescence responses depending on the optical setup used to collect the data: wide-field microscopy has resulted in a decrease in fluorescence upon cell shrinkage, whereas confocal microscopy has been shown to yield the opposite result. In this short communication, we have investigated the effect of optical setup on detection of cell volume changes in calcein-stained endothelial cells. A confocal microscope was used to collect the fluorescence data, and the pinhole diameter was varied in order to examine the effects of optical section thickness on fluorescence response. For large pinhole diameters – which correspond to relatively thick optical sections – fluorescence intensity decreased when cells were induced to shrink. In contrast, for small pinhole diameters the fluorescence intensity increased with cell shrinkage. The transition between these two types of fluorescence responses occurred when using a pinhole diameter of 285 μm, which corresponds with an optical section thickness slightly less than the height of the cells. Our results have implications for the design and interpretation of experiments involving the use of calcein as a cell volume indicator.
References
McGrath JJ (1997) Quantitative measurement of cell membrane transport: technology and applications. Cryobiology 34:315–334
Verkman AS (2000) Water permeability measurement in living cells and complex tissues. J Membr Biol 173:73–87
Verkman AS, VanHoek AN, Ma TH, Frigeri A, Skach WR, Mitra A, Tamarappoo BK, Farinas J, Van Hoek AN (1996) Water transport across mammalian cell membranes. Am J Physiol, Cell Physiol 270:C12–C30
Ruiz-Ederra J, Verkman AS (2006) Accelerated cataract formation and reduced lens epithelial water permeability in aquaporin-1-deficient mice. Invest Ophthalmol Vis Sci 47:3960–3967
Mitchell CH, Fleischhauer JC, Stamer WD, Peterson-Yantorno K, Civan MM (2002) Human trabecular meshwork cell volume regulation. Am J Physiol, Cell Physiol 283:315–326
Yellowley CE, Hancox JC, Donahue HJ (2002) Effects of cell swelling on intracellular calcium and membrane currents in bovine articular chondrocytes. J Cell Biochem 86:290–301
Zelenina M, Zelenin S, Bondar AA, Brismar H, Alexander A, Aperia A (2002) Water permeability of aquaporin-4 is decreased by protein kinase C and dopamine. Am J Physiol Renal Physiol 283:F309–F318
Altamirano J, Brodwick MS, Alvarez-Leefmans FJ (1998) Regulatory volume decrease and intracellular Ca2+ in murine neuroblastoma cells studied with fluorescent probes. J Gen Physiol 112:145–160
Capo-Aponte JE, Iserovich P, Reinach PS, Capó-Aponte JE (2005) Characterization of regulatory volume behavior by fluorescence quenching in human corneal epithelial cells. J Membr Biol 207:11–22
Crowe WE, Altamirano J, Huerto L, Alvarez-Leefmans FJ (1995) Volume changes in single N1E-115 neuroblastoma cells measured with a fluorescent probe. Neuroscience 69:283–296
Levin MH, Verkman AS (2004) Aquaporin-dependent water permeation at the mouse ocular surface: in vivo microfluorimetric measurements in cornea and conjunctiva. Invest Ophthalmol Vis Sci 45:4423–4432
Hamann S, Herrera-Perez JJ, Bundgaard M, Alvarez-Leefmans FJ, Zeuthen T (2005) Water permeability of Na+−K+−2Cl- cotransporters in mammalian epithelial cells. J Physiol (Lond) 568:123–135
Hamann S, Kiilgaard JF, Litman T, Alvarez-leefmans FJ, Winther BR, Zeuthen T (2002) Measurement of cell volume changes by fluorescence self-quenching. J Fluoresc 12:139–145
Solenov E, Watanabe H, Manley GT, Verkman AS (2004) Sevenfold-reduced osmotic water permeability in primary astrocyte cultures from AQP-4-deficient mice, measured by a fluorescence quenching method. Am J Physiol, Cell Physiol 286:426–432
Sonnentag T, Siegel WK, Bachmann O, Rossmann H, Mack A, Wagner HJ, Gregor M, Seidler U (2000) Agonist-induced cytoplasmic volume changes in cultured rabbit parietal cells. Am J Physiol Gastrointest Liver Physiol 279:G40–G48
Li L, Zhang H, Ma T, Verkman AS (2009) Very high aquaporin-1 facilitated water permeability in mouse gallbladder. Am J Physiol Gastrointest Liver Physiol 296:816–822
Zelenina M, Brismar H (2000) Osmotic water permeability measurements using confocal laser scanning microscopy. Eur Biophys J 29:165–171
Fenton RA, Moeller HB, Zelenina M, Snaebjornsson MT, Holen T, MacAulay N (2010) Differential water permeability and regulation of three aquaporin 4 isoforms. Cell Mol Life Sci 67:829–840
Fry AK, Higgins AZ (2012) Measurement of cryoprotectant permeability in adherent endothelial cells and applications to cryopreservation. Cell Mol Bioeng 5:287–298
Gao DY, Liu J, Liu C, McGann LE, Watson PF, Kleinhans FW, Mazur P, Critser ES, Critser JK (1995) Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol. Hum Reprod 10:1109–1122
Benson JD, Kearsley AJ, Higgins AZ (2012) Mathematical optimization of procedures for cryoprotectant equilibration using a toxicity cost function. Cryobiology 64:144–151
Karlsson JOM, Younis AI, Chan AWS, Gould KG, Eroglu A (2009) Permeability of the rhesus monkey oocyte membrane to water and common cryoprotectants. Mol Reprod Dev 76:321–333
Mukherjee IN, Song YC, Sambanis A (2007) Cryoprotectant delivery and removal from murine insulinomas at vitrification-relevant concentrations. Cryobiology 55:10–18
Higgins AZ, Karlsson JOM (2010) Analysis of solution exchange in flow chambers with applications to cell membrane permeability measurement. Cell Mol Bioeng 3:269–285
Higgins AZ, Karlsson JOM (2012) Comparison of cell membrane water permeability in monolayers and suspensions. Cryo-Letters 33:95–106
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Davidson, A.F., Higgins, A.Z. Detection of Volume Changes in Calcein-Stained Cells Using Confocal Microscopy. J Fluoresc 23, 393–398 (2013). https://doi.org/10.1007/s10895-013-1202-1
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DOI: https://doi.org/10.1007/s10895-013-1202-1