Name: ex vivo analysis of mechanically activated ca2+ transients in urothelial cells
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This work was supported by NIH grants R01DK119183 (to G.A. and M.D.C.) and S10OD028596 (to G.A.) and by the Cell Physiology and Model Organisms Kidney Imaging Cores of the Pittsburgh Center for Kidney Research (P30DK079307).

Care and handling of the animals were carried out in accordance with the University of Pittsburgh Institutional Animal Care and Use Committee. Female, 2-4-month-old C57Bl/6J mice were used for the present study. The mice were obtained commercially (see Table of Materials).
1. Equipment assembly and setup
<ol>
	<li>Perform Ca2+ imaging with an upright microscope equipped with a high-resolution camera and a stable light source (see Table of ...

<ol>
	<li>Kunau, R. T., Webb, H. L., Borman, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+the+relationship+between+the+flow+rate+of+tubular+fluid+and+potassium+transport+in+the+distal+tubule+of+the+rat.">Characteristics of the relationship between the flow rate of tubular fluid and potassium transport in the distal tubule of the rat.</a> Journal of Clinical Investigation. 54 (6), 1488-1495 (1974).</li><li>Engbretson, B. G., Stoner, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+potassium+secretion+by+rabbit+cortical+collecting+tubule+in+vitro.">Flow-dependent potassium secretion by rabbit cortical collecting tubule in vitro.</a> American Journal of Physiology. 253 (5), 896-903 (1987).</li><li>Satlin, L. M., Sheng, S., Woda, C. B., Kleyman, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+Na(+)+channels+are+regulated+by+flow.">Epithelial Na(+) channels are regulated by flow.</a> American Journal of Physiology Renal Physiology. 280 (6), 1010-1018 (2001).</li><li>Woda, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+flow-stimulated+potassium+secretion+in+rabbit+cortical+collecting+duct:+functional+and+molecular+aspects.">Ontogeny of flow-stimulated potassium secretion in rabbit cortical collecting duct: functional and molecular aspects.</a> American Journal of Physiology Renal Physiology. 285 (4), 629-639 (2003).</li><li>Malnic, G., Berliner, R. W., Giebisch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+dependence+of+K++secretion+in+cortical+distal+tubules+of+the+rat.">Flow dependence of K+ secretion in cortical distal tubules of the rat.</a> American Journal of Physiology. 256 (5), 932-941 (1989).</li><li>Khuri, R. N., Strieder, W. N., Giebisch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+flow+rate+and+potassium+intake+on+distal+tubular+potassium+transfer.">Effects of flow rate and potassium intake on distal tubular potassium transfer.</a> American Journal of Physiology. 228 (4), 1249-1261 (1975).</li><li>Good, D. W., Wright, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminal+influences+on+potassium+secretion:+sodium+concentration+and+fluid+flow+rate.">Luminal influences on potassium secretion: sodium concentration and fluid flow rate.</a> American Journal of Physiology. 236 (2), 192-205 (1979).</li><li>Wong, K. R., Berry, C. A., Cogan, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+dependence+of+chloride+transport+in+rat+S1+proximal+tubules.">Flow dependence of chloride transport in rat S1 proximal tubules.</a> American Journal of Physiology. 269 (6), 870-875 (1995).</li><li>Garvin, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+absorption+by+isolated+perfused+rat+proximal+straight+tubules.">Glucose absorption by isolated perfused rat proximal straight tubules.</a> American Journal of Physiology. 259 (4), 580-586 (1990).</li><li>Malnic, G., Klose, R. M., Giebisch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropuncture+study+of+renal+potassium+excretion+in+the+rat.">Micropuncture study of renal potassium excretion in the rat.</a> American Journal of Physiology. 206 (4), 674-686 (1964).</li><li>Malnic, G., Klose, R. M., Giebisch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropuncture+study+of+distal+tubular+potassium+and+sodium+transport+in+rat+nephron.">Micropuncture study of distal tubular potassium and sodium transport in rat nephron.</a> American Journal of Physiology. 211 (3), 529-547 (1966).</li><li>Cabral, P. D., Garvin, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminal+flow+regulates+NO+and+O2(-)+along+the+nephron.">Luminal flow regulates NO and O2(-) along the nephron.</a> American Journal of Physiology. 300 (5), 1047-1053 (2011).</li><li>Raghavan, V., Rbaibi, Y., Pastor-Soler, N. M., Carattino, M. D., Weisz, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress-dependent+regulation+of+apical+endocytosis+in+renal+proximal+tubule+cells+mediated+by+primary+cilia.">Shear stress-dependent regulation of apical endocytosis in renal proximal tubule cells mediated by primary cilia.</a> Proceedings of the National Academy of Sciences. 111 (23), 8506-8511 (2014).</li><li>Lewis, S. A., de Moura, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical+membrane+area+of+rabbit+urinary+bladder+increases+by+fusion+of+intracellular+vesicles:+an+electrophysiological+study.">Apical membrane area of rabbit urinary bladder increases by fusion of intracellular vesicles: an electrophysiological study.</a> The Journal of Membrane Biology. 82 (2), 123-136 (1984).</li><li>Fowler, C. J., Griffiths, D., de Groat, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+control+of+micturition.">The neural control of micturition.</a> Nature Reviews Neuroscience. 9 (6), 453-466 (2008).</li><li>Dalghi, M. G., Montalbetti, N., Carattino, M. D., Apodaca, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+urothelium:+life+in+a+liquid+environment.">The urothelium: life in a liquid environment.</a> Physiological Reviews. 100 (4), 1621-1705 (2020).</li><li>Khandelwal, P., Abraham, S. N., Apodaca, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+and+physiology+of+the+uroepithelium.">Cell biology and physiology of the uroepithelium.</a> American Journal of Physiology Renal Physiology. 297 (6), 1477-1501 (2009).</li><li>Sachs, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch-activated+ion+channels:+what+are+they.">Stretch-activated ion channels: what are they.</a> Physiology. 25 (1), 50-56 (2010).</li><li>Martinac, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+ion+channels:+molecules+of+mechanotransduction.">Mechanosensitive ion channels: molecules of mechanotransduction.</a> Journal of Cell Science. 117 (12), 2449-2460 (2004).</li><li>Ranade, S. S., Syeda, R., Patapoutian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanically+activated+ion+channels.">Mechanically activated ion channels.</a> Neuron. 87 (6), 1162-1179 (2015).</li><li>Cox, C. D., Bavi, N., Martinac, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+principles+of+ion-channel-mediated+mechanosensory+transduction.">Biophysical principles of ion-channel-mediated mechanosensory transduction.</a> Cell Reports. 29 (1), 1-12 (2019).</li><li>Carattino, M. D., Sheng, S., Kleyman, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+Na++channels+are+activated+by+laminar+shear+stress.">Epithelial Na+ channels are activated by laminar shear stress.</a> Journal of Biological Chemistry. 279 (6), 4120-4126 (2004).</li><li>Ross, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+in+mechanotransduction.">Integrins in mechanotransduction.</a> Current Opinion in Cell Biology. 25 (5), 613-618 (2013).</li><li>Dieterle, M. P., Husari, A., Rolauffs, B., Steinberg, T., Tomakidi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins,+cadherins+and+channels+in+cartilage+mechanotransduction:+perspectives+for+future+regeneration+strategies.">Integrins, cadherins and channels in cartilage mechanotransduction: perspectives for future regeneration strategies.</a> Expert Reviews in Molecular Medicine. 23, 14 (2021).</li><li>Huveneers, S., de Rooij, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+systems+at+the+cadherin-F-actin+interface.">Mechanosensitive systems at the cadherin-F-actin interface.</a> Journal of Cell Science. 126, 403-413 (2013).</li><li>Sun, Z., Guo, S. S., F&#228;ssler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-mediated+mechanotransduction.">Integrin-mediated mechanotransduction.</a> Journal of Cell Biology. 215 (4), 445-456 (2016).</li><li>Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.+Pfl&#252;gers+Archiv.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfl&#252;gers Archiv.</a> European Journal of Physiology. 391 (2), 85-100 (1981).</li><li>Akerboom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+GCaMP+calcium+indicator+for+neural+activity+imaging.">Optimization of a GCaMP calcium indicator for neural activity imaging.</a> The Journal of Neuroscience. 32 (40), 13819-13840 (2012).</li><li>Akerboom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+calcium+indicators+for+multi-color+neural+activity+imaging+and+combination+with+optogenetics.">Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics.</a> Frontiers in Molecular Neuroscience. 6, 2 (2013).</li><li>Sun, X. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+GCaMPs+for+improved+tracking+of+neuronal+activity.">Fast GCaMPs for improved tracking of neuronal activity.</a> Nature Communications. 4, 2170 (2013).</li><li>Dalghi, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+roles+for+PIEZO1+and+PIEZO2+in+urothelial+mechanotransduction+and+lower+urinary+tract+interoception.">Functional roles for PIEZO1 and PIEZO2 in urothelial mechanotransduction and lower urinary tract interoception.</a> JCI Insight. 6 (19), 152984 (2021).</li><li>Durnin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+bladder+model+with+detrusor+smooth+muscle+removed+to+analyse+biologically+active+mediators+released+from+the+suburothelium.">An ex vivo bladder model with detrusor smooth muscle removed to analyse biologically active mediators released from the suburothelium.</a> The Journal of Physiology. 597 (6), 1467-1485 (2019).</li><li>Delmas, P., Coste, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechano-gated+ion+channels+in+sensory+systems.">Mechano-gated ion channels in sensory systems.</a> Cell. 155 (2), 278-284 (2013).</li><li>Tavernarakis, N., Driscoll, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degenerins.+At+the+core+of+the+metazoan+mechanotransducer.">Degenerins. At the core of the metazoan mechanotransducer.</a> Annals of the New York Academy of Sciences. 940 (1), 28-41 (2001).</li><li>Peyronnet, R., Tran, D., Girault, T., Frachisse, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+channels:+feeling+tension+in+a+world+under+pressure.">Mechanosensitive channels: feeling tension in a world under pressure.</a> Frontiers in Plant Science. 5, 558 (2014).</li><li>Blount, P., Iscla, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+with+bacterial+mechanosensitive+channels,+from+discovery+to+physiology+to+pharmacological+target.">Life with bacterial mechanosensitive channels, from discovery to physiology to pharmacological target.</a> Microbiology and Molecular Biology Reviews. 84 (1), 00055 (2020).</li><li>Booth, I. R., Miller, S., M&#252;ller, A., Lehtovirta-Morley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+bacterial+mechanosensitive+channels.">The evolution of bacterial mechanosensitive channels.</a> Cell Calcium. 57 (3), 140-150 (2015).</li></ol>

All organisms, and seemingly most cell types, express ion channels that respond to mechanical stimuli20,33,34,35,36,37. The function of these mechanically activated channels has been predominantly assessed with the patch-clamp technique. However, due to accessibility issues, patch-clamp studies of mechanically activated ion c...

Epithelial cells in the urinary tract are subjected to mechanical forces as the urinary filtrate travels through the nephrons, and urine is pumped out of the renal pelvis and travels through the ureters to be stored in the urinary bladder. It has been long recognized that mechanical forces (e.g., shear stress and stretch) exerted by fluids on the epithelial cells that line the urinary tract regulate the reabsorption of protein in the proximal tubule and of solutes in the distal nephron1,2,3,4,5,6,7,8,9,10,11,12,13, as well as the storage of urine in the urinary bladder and micturition14,15,16,17.
The conversion of mechanical stimuli into electrical and biochemical signals, a process referred to as mechanotransduction, is mediated by proteins that respond to the deformation of cellular structures or the associated extracellular matrix18,19,20,21. Mechanically activated ion channels are unique in the sense that they transition from a closed state to an open permeable state in response to changes in membrane tension, pressure, or shear stress18,19,20,21,22. In addition, Ca2+ transients can be initiated by&#160;integrin-mediated mechanotransduction or by activation of mechanoresponsive adhesion systems at cell-cell junctions23,24,25,26. Ion channel function is usually assessed with the patch-clamp technique, which involves the formation of a gigaohm seal between the cell membrane and the patch pipette27. However, cells located in deep tissue layers with a dense extracellular matrix (e.g., kidney tubules) or surrounded by a physical barrier (e.g., glycocalyx) are difficult to access with a glass micropipette. Likewise, cells embedded or that are integral parts of tissues with poor mechanical stability (e.g., the urothelium) can not be readily studied with the patch-clamp technique. Because many mechanically activated ion channels are permeable to Ca2+, an alternative approach is to assess their activity by fluorescent microscopy using a Ca2+-sensitive dye or genetically encoded calcium indicators (GECIs) such as GCaMP. Recent efforts in protein engineering have significantly increased the dynamic range, sensitivity, and response of GECIs28,29,30, and advances in genetics have allowed their expression in specific cell populations, making them ideally suited to study mechanotransduction.
The urothelium, the stratified epithelium that covers the interior of the urinary bladder, functions as a barrier, preventing the diffusion of urinary solutes into the bladder interstitium, but also functions as a transducer, sensing bladder fullness and communicating these events to the underlying nerves and musculature16. Previous studies have shown that the communication between the urothelium and underlying tissues requires the mechanically activated ion channels Piezo1 and Piezo231. To assess mechanically induced Ca2+ transients in urothelial cells, a new technique described that uses adenoviral gene transfer to express the Ca2+ sensor GCaMP5G in urothelial cells was developed. This technique employs a mucosal sheet preparation that provides easy access to the outermost umbrella cell layer and a computer-assisted system for the simultaneous mechanical stimulation of individual cells with a closed glass micropipette and recording of changes in fluorescence over time.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20x Objective</td>
			<td>Olympus</td>
			<td>UMPlanFL N</td>
			<td></td>
		</tr>
		<tr>
			<td>24 G &#190;&#8221; catheter</td>
			<td>Medline&#160;</td>
			<td>Suresite IV slide&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>4x Objective</td>
			<td>Olympus</td>
			<td>UPlanFL N</td>
			<td></td>
		</tr>
		<tr>
			<td>Analog/digital converter</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Abcam&#160;</td>
			<td>Ab6556</td>
			<td></td>
		</tr>
		<tr>
			<td>Beam splitter</td>
			<td>Chroma</td>
			<td>T495lpxr</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar temperature controller&#160;</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fluka</td>
			<td>21114-1L</td>
			<td>1 M solution</td>
		</tr>
		<tr>
			<td>cellSens software</td>
			<td>Olympus</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>CMOS camera</td>
			<td>Hamamatsu</td>
			<td>ORCA fusion</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit conjugated to Alexa Fluor 488&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-545-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter&#160;</td>
			<td>Chroma&#160;</td>
			<td>ET470/40X</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillaries Corning 8250 glass</td>
			<td>Warner Instruments&#160;</td>
			<td>G85150T-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline heater&#160;</td>
			<td>Warner Instruments</td>
			<td>SH-27B</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>793590</td>
			<td></td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Sutter Instruments</td>
			<td>Lambda XL&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Manifold pump tubing</td>
			<td>Fisherbrand</td>
			<td>14-190-510</td>
			<td>ID 1.52 mm</td>
		</tr>
		<tr>
			<td>Manifold pump tubing</td>
			<td>Fisherbrand</td>
			<td>14-190-533</td>
			<td>ID 2.79 mm</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice&#160;</td>
			<td>Jackson Lab</td>
			<td>664</td>
			<td>2-4 months old female C57BL/6J</td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige&#160;</td>
			<td>MF-830</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MP-285</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51W</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media with DAPI</td>
			<td>Invitrogen</td>
			<td>S36964&#160;</td>
			<td>Slowfade Diamond Antifade with DAPI</td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>pClamp software</td>
			<td>Molecular Devices</td>
			<td>Version 10.4</td>
			<td>Patch-clamp electrophysiology data acquisition and analysis software</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson</td>
			<td>Minipuls 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezoelectric actuator</td>
			<td>Thorlabs</td>
			<td>PAS005</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette holder</td>
			<td>World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Narishige</td>
			<td>PP-830</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick exchange heated base with perfusion and adapter ring kit</td>
			<td>Warner Instruments</td>
			<td>QE-1</td>
			<td>Quick exchange platform fits 35 mm dish&#160;&#160;</td>
		</tr>
		<tr>
			<td>Rhodamine-phalloidin&#160;</td>
			<td>Invitrogen</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma-Plot</td>
			<td>Systat Software Inc</td>
			<td>Version 14.0</td>
			<td>Scientific graphing and data analysis software&#160;&#160;</td>
		</tr>
		<tr>
			<td>Silicone elastomer</td>
			<td>Dow</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel open-loop piezo controller</td>
			<td>Thorlabs</td>
			<td>MDT694B</td>
			<td></td>
		</tr>
		<tr>
			<td>Square grid holder pad</td>
			<td>Ted Pella</td>
			<td>10520</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>AD Surgical</td>
			<td>S-S618R13</td>
			<td>6-0 Sylk</td>
		</tr>
		<tr>
			<td>Teflon mounting rod</td>
			<td>Custom made</td>
			<td></td>
			<td>Use to mount the piezoelectric actuator in the micromanipulator</td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>Fisher Scientific</td>
			<td>14171129</td>
			<td>Tygon S3 ID 1/16 IN, OD 1/8 IN</td>
		</tr>
		<tr>
			<td>USB Digital I/O device&#160;</td>
			<td>National Instruments</td>
			<td>NI USB-6501</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo analysis of mechanically activated ca2+ transients in urothelial cells

Mechanically activated ion channels are biological transducers that convert mechanical stimuli such as stretch or shear forces into electrical and biochemical signals. In mammals, mechanically activated channels are essential for the detection of external and internal stimuli in processes as diverse as touch sensation, hearing, red blood cell volume regulation, basal blood pressure regulation, and the sensation of urinary bladder fullness. While the function of mechanically activated ion channels has been extensively studied in the in vitro setting using the patch-clamp technique, assessing their function in their native environment remains a difficult task, often because of limited access to the sites of expression of these channels (e.g., afferent terminals, Merkel cells, baroreceptors, and kidney tubules) or difficulties applying the patch-clamp technique (e.g., the apical surfaces of urothelial umbrella cells). This protocol describes a procedure to assess mechanically evoked Ca2+ transients using the fluorescent sensor GCaMP5G in an ex vivo urothelial preparation, a technique that could be readily adapted for the study of mechanically evoked Ca2+ events in other native tissue preparations.

The present protocol describes a technique to assess mechanically evoked Ca2+ transients in umbrella cells using the fluorescent Ca2+ sensor GCaMP5G. Adenoviral transduction was employed to express GCaMP5G in urothelial cells due to its high efficiency and because it produces an elevated level of expression. Fluorescent images of stained cryosections from a transduced bladder are shown in Figure 2D. For these experiments, GCaMP5G expression is highest in the umbrella ce...

Watch this Scientific Journal Video about Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells at JoVE.com

Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells

This protocol describes a methodology to assess the function of mechanically activated ion channels in native urothelial cells using the fluorescent Ca2+ sensor GCaMP5G.

Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells

Mechanically activated ion channels are biological transducers that convert mechanical stimuli such as stretch or shear forces into electrical and ...

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