Research in the laboratories is funded by the Fritz-Bender Foundation, Munich, Germany (to T.D.) and a University of Witten/Herdecke internal research grant (to W.-K.L). We would like to thank Prof Dr. Dr. Frank Th&#233;venod (Institute for Physiology, Pathophysiology &#38; Toxicology, University of Witten/Herdecke) for helpful suggestions.

1. Preparation of Reagents and Solutions
<ol>
	<li>Prepare a modified Hank&#8217;s balanced salt solution (HBSS) (in mM: 136.9 NaCl, 5.37 KCl, 0.34 Na2HPO4, 0.44 KH2PO4, 4.17 NaHCO3, pH 7.2, no phenol red). Store at 4 &#176;C.</li>
	<li>Make 1.5 M CaCl2 and 2 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.2) stock solutions using distilled water.</li>
	<li>Prepare a 250 mM stock solution of probenecid. For the free acid form (often re...

<ol>
	<li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat Rev Mol Cell Biol. 1, 11-21 (2000).</li><li>Brini, M., Carafoli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+pumps+in+health+and+disease.">Calcium pumps in health and disease.</a> Physiol Rev. 89, 1341-1378 (2009).</li><li>Berridge, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+trisphosphate+and+calcium+signalling.">Inositol trisphosphate and calcium signalling.</a> Nature. 361, 315-325 (1993).</li><li>Putney, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+actions+of+calcium-mobilizing+messenger,+inositol+1,4,5-trisphosphate.">Formation and actions of calcium-mobilizing messenger, inositol 1,4,5-trisphosphate.</a> Am J Physiol. 252, 149-157 (1987).</li><li>Putney, J. W., Ribeiro, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+between+the+plasma+membrane+and+endoplasmic+reticulum+calcium+stores.">Signaling pathways between the plasma membrane and endoplasmic reticulum calcium stores.</a> Cell Mol Life Sci. 57, 1272-1286 (2000).</li><li>Soboloff, J., Rothberg, B. S., Madesh, M., Gill, D. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> J Biol Chem. 260, 3440-3450 (1985).</li><li>Jin, X., 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=Cilioplasm+is+a+cellular+compartment+for+calcium+signaling+in+response+to+mechanical+and+chemical+stimuli.">Cilioplasm is a cellular compartment for calcium signaling in response to mechanical and chemical stimuli.</a> Cell Mol Life Sci. , (2013).</li><li>Valet, G., Raffael, A., Russmann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+intracellular+calcium+in+vital+cells+by+flow-cytometry.">Determination of intracellular calcium in vital cells by flow-cytometry.</a> Naturwissenschaften. 72, 600-602 (1985).</li><li>June, C. H., Rabinovitch, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+measurement+of+cellular+ionized+calcium+concentration.">Flow cytometric measurement of cellular ionized calcium concentration.</a> Pathology and immunopathology research. 7, 409-432 (1988).</li><li>Ransom, J. T., DiGiusto, D. L., Cambier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+intracellular+calcium+mobilization.">Flow cytometric analysis of intracellular calcium mobilization.</a> Methods Enzymol. 141, 53-63 (1987).</li><li>Gergely, L., Cook, L., Agnello, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+Ca2++flux+measurement+on+isolated+human+B+cells+that+uses+flow+cytometry.">A simplified method for Ca2+ flux measurement on isolated human B cells that uses flow cytometry.</a> Clin Diagn Lab Immunol. 4, 70-74 (1997).</li><li>Seidel, 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=The+neurotransmitter+GABA+is+a+potent+inhibitor+of+the+stromal+cell-derived+factor-1alpha+induced+migration+of+adult+CD133++hematopoietic+stem+and+progenitor+cells.">The neurotransmitter GABA is a potent inhibitor of the stromal cell-derived factor-1alpha induced migration of adult CD133+ hematopoietic stem and progenitor cells.</a> Stem cells and development. 16, 827-836 (2007).</li><li>Lee, W. K., Chakraborty, P. K., Roussa, E., Wolff, N. A., Th&#233;venod, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERK1/2-dependent+bestrophin-3+expression+prevents+ER-stress-induced+cell+death+in+renal+epithelial+cells+by+reducing+CHOP.">ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP.</a> Biochim Biophys Acta. 1823, 1864-1876 (2012).</li><li>Woost, P. 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=Immortalization+and+characterization+of+proximal+tubule+cells+derived+from+kidneys+of+spontaneously+hypertensive+and+normotensive+rats.">Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats.</a> Kidney Int. 50, 125-134 (1996).</li><li>Beyer, K. H., 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=Benemid,'+p-(di-n-propylsulfamyl)-benzoic+acid;+its+renal+affinity+and+its+elimination.">Benemid,' p-(di-n-propylsulfamyl)-benzoic acid; its renal affinity and its elimination.</a> Am J Physiol. 166, 625-640 (1951).</li><li>Buckley, B. J., Whorton, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunicamycin+increases+intracellular+calcium+levels+in+bovine+aortic+endothelial+cells.">Tunicamycin increases intracellular calcium levels in bovine aortic endothelial cells.</a> Am J Physiol. 273, 1298-1305 (1997).</li><li>Koepsell, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SLC22+family+with+transporters+of+organic+cations,+anions+and+zwitterions.">The SLC22 family with transporters of organic cations, anions and zwitterions.</a> Molecular aspects of medicine. 34, 413-435 (2013).</li><li>Dean, M., Hamon, Y., Chimini, 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+human+ATP-binding+cassette+(ABC)+transporter+superfamily.">The human ATP-binding cassette (ABC) transporter superfamily.</a> J Lipid Res. 42, 1007-1017 (2001).</li><li>Brezden, C. 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Calcium is a key event in multiple cellular processes acting as a second messenger signaling molecule and also as a means to communicate between the ER and mitochondria. The ability to measure changes in free cytosolic calcium concentrations is a useful technique that can apply to all areas of cell biology. There are various methods to detect cytosolic calcium signals. Classically, signals from a ratiometric calcium indicator, such as Fura-2, are monitored in live cells using a fluorescence imaging setup and absolute cal...

Calcium is an important second messenger responsible for the transmission of cellular physiological and pathophysiological signals1. Cytosolic calcium concentrations are kept low through the activity of calcium pumps and calcium exchangers. The sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) refills the endoplasmic reticulum (ER) calcium store as part of a &#8220;pump leak&#8221; system whereas the plasma membrane calcium ATPase (PMCA) extrudes calcium to the extracellular compartment, both in ATP-dependent manners2. Physiological messengers, such as hormones or neurotransmitters, transmit their signals through G-protein coupled receptors, activation of phospholipase C, which hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane to generate diacylglycerol and inositol triphosphate (IP3)3. While diacylglycerol remains in the plasma membrane, IP3 diffuses into the cytosol and binds to IP3 receptors (IP3Rs), which are ligand activated calcium channels, found in the ER membrane and calcium is released from the ER luminal store culminating in an increase in cytosolic calcium concentrations4. An alternative route for calcium to reach the cytosol is through calcium channels and exchangers present in the plasma membrane. Crosstalk between these two compartments have been described: calcium induced calcium release (CICR) where extracellular calcium induces calcium release from ER stores5, and store operated calcium entry (SOCE) where emptying of the ER stores is sensed by STIM and causes opening of Orai calcium channels in the plasma membrane to promote refilling of the ER stores6.
Under pathophysiological conditions, the cellular calcium response is deregulated and cytosolic calcium increases in the absence of physiological stimulators1. The calcium response may be affected in a number of ways: prolonged calcium signal, delayed calcium removal from the cytosol, depletion of calcium stores or localized changes in calcium. Furthermore, the mitochondria take on a central role in buffering and releasing calcium7. Prolonged and/or supramaximal cytosolic calcium increase leads to cell death. In fact, calcium is more often than not involved in the cellular pathophysiological response and is a key event in a wide array of diseases, such as neurodegenerative disease, heart disease, cancer, bone disease and kidney disease, which are mainly associated with cell death and loss or alteration of organ or tissue function8-10. Moreover, perturbation of calcium signals has been linked to cellular adaptation and changes in cellular functions and responses.
Traditionally, calcium signals are measured with a negatively charged fluorescent calcium indicator that is loaded into cells in a cell permeable acetoxymethyl (AM) ester form. Once cleaved by cellular esterases, the fluorescent indicators remain within the intracellular compartment and increase in fluorescence intensity when calcium is bound. The most well known and used calcium indicator is the ratiometric Fura-2 developed by Roger Tsien and colleagues11. Calcium indicators with differing affinities for calcium allow different calcium pools to be monitored. Detection methods include live cell imaging, fluorescence microplate reader and flow cytometry. The relatively fast kinetics of calcium signals (usually within a few seconds to minutes) makes live cell imaging the best method for gaining the most information about characteristics of the calcium signal. Aside from the fast kinetics of calcium signals (within milliseconds), live cell imaging is a better method for studying cellular compartmentalization of calcium signaling within a single cell 12. Absolute calcium concentrations can be calculated by determining the minimum and maximum fluorescence of the calcium indicator by adding a calcium chelator and permeabilizing the cells, respectively, as described by Grynkiewicz et al.11.
The use of flow cytometry to measure calcium signals was first developed in the 1980s in immunological cells where the opportunity to measure multiple parameters, such as membrane integrity and separation of cell population, and the requirement of cells in suspension combined with the development of single wavelength calcium indicators made flow cytometry an ideal and convenientdetection method13-15. In adherent cells, live cell imaging provides the most information about calcium signaling, most importantly the kinetics, but requires a complicated setup comprising a fluorescence microscope, a perfusion system, maintenance of the cellular environment, such as temperature, and specialized microscope software for acquiring and analyzing data. Alternative methods such as a fluorescence microplate reader or pharmacological means through use of calcium chelators are incomparable in terms of gained information. Flow cytometry is becoming more widely used to monitor cytosolic calcium in adherent cells though, in the majority of studies, only end-point measurements are performed. Using the method initially developed by Gergely et al. in immunological B cells16, changes in cytosolic free calcium concentration by flow cytometry with real-time kinetics and monitoring the fluorescence intensity in individual cells from a sample population have been successfully measured in cancer epithelial cells and cancer stem cell hybrids17. This method was further adapted for use in adherent epithelial cells, which are difficult to load with calcium indicators 18.
Here, using an immortalized adherent epithelial cell line derived from the S1 segment of the rat kidney proximal tubule (WKPT-0293 Cl.2)19, we describe an optimized simplified method for measurement of changes in cytosolic calcium concentration by flow cytometry. Since many epithelial cells possess a number of organic transporters for anionic compounds, loading of cells with a calcium indicator can prove to be a challenge. To prevent the efflux of calcium indicators, probenecid, the prototypical inhibitor of organic anion transporters and originally developed to decrease renal excretion of penicillin20, is applied simultaneously in the incubation medium. The cells are maintained in monolayers for calcium indicator loading, brought into suspension and calcium signals can be detected immediately after compound addition.

<table border="0" cellpadding="0" cellspacing="0" width="735">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:79px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:321px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-3, AM</td>
			<td style="width:79px;">Invitrogen</td>
			<td style="width:119px;">F-1241&#160;</td>
			<td>Cell permeable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Probenecid</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-8761</td>
			<td>Water insoluble, dissolve in 1 N NaOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.05% Trypsin-EDTA (1X)</td>
			<td style="width:79px;">Invitrogen</td>
			<td style="width:119px;">25300-062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ionomycin</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">I9657</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thapsigargin</td>
			<td style="width:79px;">Tocris Bioscience</td>
			<td style="width:119px;">1138</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tunicamycin</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">T7765</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACSCalibur Flow Cytometer + CELLQuest software</td>
			<td style="width:79px;">Becton Dickinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Windows Multiple Document Interface for Flow Cytometry (WinMDI)</td>
			<td style="width:79px;">Joe Trotter, The Scripps Institute</td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Please note that this is an older 16-bit application that reads FCS 2.0 compliant files but does not recognize FCS 3.0 digital data.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">WKPT-0293 Cl.2 rat kidney proximal tubule cell line</td>
			<td style="width:79px;"></td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Made available by Dr. Ulrich Hopfer (Department of Physiology &#38; Biophysics, Case 
			Western Reserve University, Cleveland, OH)</td>
		</tr>
	</tbody>
</table>

cytosolic calcium measurements in renal epithelial cells by flow cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

To increase cytosolic calcium, two pharmacological compounds were applied to renal proximal tubule cells (WKPT-0293 Cl.2) loaded with cell permeable calcium binding dye, Fluo-3-AM. Nontreated control samples were loaded with calcium binding dye in the presence of probenecid and were mixed but without the addition of any compounds. Thapsigargin (TG) is a classical inhibitor of the SERCA pump leading to leakage out of the ER and resulting in increased cytosolic calcium. Tunicamycin (TUN) blocks N-glycosylation of proteins ...

Watch this Scientific Journal Video about Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry at JoVE.com

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

cytosolic-calcium-measurements-renal-epithelial-cells-flow

Research

JoVE Journal

Biology

15.1K Aufrufe.  University of Witten/Herdecke.  Calcium ist in vielen physiologischen und pathophysiologischen Signalwegen beteiligt sind. Live Cell Imaging erfordert spezielle Geräte und kann zeitaufwendig sein. Eine schnelle, einfache Methode mit einem Durchflusszytometer, um relative Veränderungen in zytosolischen Kalzium in adhärenten Epithelzellen in Suspension gebracht bestimmen optimiert. 