Name: monitoring er/sr calcium release with the targeted ca2+ sensor catcher+
Uploaded: 2017-05-19T15:00:00
Description: Watch this Scientific Journal Video about Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+ at JoVE.com

This work was funded by NIH GM62999, NIH EB007268, NIH AG15820, B&amp;B Seed Grant, and a NIH Supplemental Grant to FR, BB fellowship to CM, CDT fellowship to RG.

1. Slide Preparation
<ol>
	<li>Place 22 mm x 40 mm glass microscope slides in 6 cm cell culture dishes, 1 slide per dish.</li>
	<li>Expose each side of each slide as well as the 6 cm dish to ultraviolet (UV) light for 15-20 min to sterilize in a sterile hood.</li>
	<li>Cover the dishes with slides inside with parafilm and store at 4 &#176;C until ready to use.</li>
</ol>
2. Preparation of Media, Buffers, Solutions, and Reagents
<ol>
	<li>Prepare 1 L of Hank&#39;s balanced salt solution (HBSS) ...

<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 (1), 11-21 (2000).</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=Calcium+signalling+remodelling+and+disease.">Calcium signalling remodelling and disease.</a> Biochem. Soc. Trans. 40, 297-309 (2012).</li><li>Tang, S., Reddish, F., Zhuo, Y., Yang, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+kinetics+of+calcium+signaling+and+sensor.">Fast kinetics of calcium signaling and sensor.</a> Curr. Opin. Chem. Biol. 27, 90-97 (2015).</li><li>Rizzuto, R., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdomains+of+intracellular+Ca2+:+Molecular+determinants+and+functional+consequences.">Microdomains of intracellular Ca2+: Molecular determinants and functional consequences.</a> Physiol. Rev. 86 (1), 369-408 (2006).</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=The+endoplasmic+reticulum:+a+multifunctional+signaling+organelle.">The endoplasmic reticulum: a multifunctional signaling organelle.</a> Cell Calcium. 32 (5-6), 235-249 (2002).</li><li>Clapham, D. 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+Signaling.">Calcium Signaling.</a> Cell. 131 (6), 1047-1058 (2007).</li><li>HerrmannFrank, A., Richter, M., Sarkozi, S., Mohr, U., LehmannHorn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-chloro-m-cresol,+a+potent+and+specific+activator+of+the+skeletal+muscle+ryanodine+receptor.">4-chloro-m-cresol, a potent and specific activator of the skeletal muscle ryanodine receptor.</a> Biochimica Et Biophysica Acta-General Subjects. 1289 (1), 31-40 (1996).</li><li>Ferris, C. D., Huganir, R. L., Bredt, D. S., Cameron, A. M., Snyder, S. H. <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+receptor:+phosphorylation+by+protein+kinase+C+and+calcium+calmodulin-dependent+protein+kinases+in+reconstituted+lipid+vesicles.">Inositol trisphosphate receptor: phosphorylation by protein kinase C and calcium calmodulin-dependent protein kinases in reconstituted lipid vesicles.</a> Proc. Natl. Acad. Sci. 88 (6), 2232-2235 (1991).</li><li>Dubyak, G. R., el-Moatassim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+via+P2-purinergic+receptors+for+extracellular+ATP+and+other+nucleotides.">Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides.</a> Am. J. Physiol., Cell Physiol. 265 (3 Pt 1), C577-C606 (1993).</li><li>Song, Z., Vijayaraghavan, S., Sladek, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+increases+intracellular+calcium+in+supraoptic+neurons+by+activation+of+both+P2X+and+P2Y+purinergic+receptors.">ATP increases intracellular calcium in supraoptic neurons by activation of both P2X and P2Y purinergic receptors.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 292 (1), R423-R431 (2007).</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 (4), 1341-1378 (2009).</li><li>Michelangeli, F., East, 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=A+diversity+of+SERCA+Ca2++pump+inhibitors.">A diversity of SERCA Ca2+ pump inhibitors.</a> Biochem. Soc. Trans. 39 (3), 789-797 (2011).</li><li>Hofer, A. M., Landolfi, B., Debellis, L., Pozzan, T., Free Curci, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++dynamics+measured+in+agonist-sensitive+stores+of+single+living+intact+cells:+a+new+look+at+the+refilling+process.">Ca2+ dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process.</a> Embo J. 17 (7), 1986-1995 (1998).</li><li>Hofer, A. M., Machen, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+For+InSitu+Measurement+of+Calcium+in+Intracellular+Inositol+1,4,5-Trisphosphate-Sensitive+Stores+Using+The+Fluorescent+Indicator+Mag-Fura-2.">Technique For InSitu Measurement of Calcium in Intracellular Inositol 1,4,5-Trisphosphate-Sensitive Stores Using The Fluorescent Indicator Mag-Fura-2.</a> Proc. Natl. Acad. Sci. U. S. A. 90 (7), 2598-2602 (1993).</li><li>Raju, B., Murphy, E., Levy, L. A., Hall, R. D., London, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescent+indicator+for+measuring+cytosolic+free+magnesium.">A fluorescent indicator for measuring cytosolic free magnesium.</a> Am J Physiol. 256, (1989).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu. Rev. Biochem. 67, 509-544 (1998).</li><li>Miyawaki, A., 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=Fluorescent+indicators+for+Ca2++based+on+green+fluorescent+proteins+and+calmodulin.">Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.</a> Nature. 388 (6645), 882-887 (1997).</li><li>Palmer, A. E., Jin, C., Reed, J. C., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-2-mediated+alterations+in+endoplasmic+reticulum+Ca2++analyzed+with+an+improved+genetically+encoded+fluorescent+sensor.">Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor.</a> Proc. Natl. Acad. Sci. U. S. A. 101 (50), 17404-17409 (2004).</li><li>Heim, N., Griesbeck, O. <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+Indicators+of+Cellular+Calcium+Dynamics+Based+on+Troponin+C+and+Green+Fluorescent+Protein.">Genetically Encoded Indicators of Cellular Calcium Dynamics Based on Troponin C and Green Fluorescent Protein.</a> J. Biol. Chem. 279 (14), 14280-14286 (2004).</li><li>Mank, M., 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=A+FRET-based+calcium+biosensor+with+fast+signal+kinetics+and+high+fluorescence+change.">A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.</a> Biophys. J. 90 (5), 1790-1796 (2006).</li><li>Garaschuk, O., Griesbeck, O., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troponin+C-based+biosensors:+A+new+family+of+genetically+encoded+indicators+for+in+vivo+calcium+imaging+in+the+nervous+system.">Troponin C-based biosensors: A new family of genetically encoded indicators for in vivo calcium imaging in the nervous system.</a> Cell Calcium. 42 (4-5), 351-361 (2007).</li><li>Carlson, H. J., Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+Analysis+of+a+Red+Fluorescent+Protein-Based+Calcium+Ion+Indicator.">Mutational Analysis of a Red Fluorescent Protein-Based Calcium Ion Indicator.</a> Sensors. 13 (9), 11507-11521 (2013).</li><li>Wang, Q., Shui, B., Kotlikoff, M. I., Sondermann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Basis+for+Calcium+Sensing+by+GCaMP2.">Structural Basis for Calcium Sensing by GCaMP2.</a> Structure. 16 (12), 1817-1827 (2008).</li><li>Koldenkova, V. P., Nagai, T. <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+Ca2++indicators:+Properties+and+evaluation.">Genetically encoded Ca2+ indicators: Properties and evaluation.</a> Biochim. Biophys. Acta-Mol. Cell Res. 1833 (7), 1787-1797 (2013).</li><li>Tang, S., 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=Design+and+application+of+a+class+of+sensors+to+monitor+Ca2++dynamics+in+high+Ca2++concentration+cellular+compartments.">Design and application of a class of sensors to monitor Ca2+ dynamics in high Ca2+ concentration cellular compartments.</a> Proc. Natl. Acad. Sci. U. S. A. 108 (39), 16265-16270 (2011).</li><li>Zou, 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=Developing+sensors+for+real-time+measurement+of+high+Ca2++concentrations.">Developing sensors for real-time measurement of high Ca2+ concentrations.</a> Biochemistry. 46 (43), 12275-12288 (2007).</li><li>Wang, Z. -. M., Tang, S., Messi, M. L., Yang, J. J., Delbono, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+sarcoplasmic+reticulum+Ca2++concentration+after+Ca2++release+in+skeletal+myofibers+from+young+adult+and+old+mice.">Residual sarcoplasmic reticulum Ca2+ concentration after Ca2+ release in skeletal myofibers from young adult and old mice.</a> Pflugers Arch., EJP. 463 (4), 615-624 (2012).</li><li>Pedelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C., Waldo, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+characterization+of+a+superfolder+green+fluorescent+protein.">Engineering and characterization of a superfolder green fluorescent protein.</a> Nat. Biotechnol. 24 (1), 79-88 (2006).</li><li>Siemering, K. R., Golbik, R., Sever, R., Haseloff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+that+suppress+the+thermosensitivity+of+green+fluorescent+protein.">Mutations that suppress the thermosensitivity of green fluorescent protein.</a> Current Biology. 6 (12), 1653-1663 (1996).</li><li>Reddish, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Design Of Genetically-Encoded Ca2+ Probes With Rapid Kinetics for Sub-Cellular Application [dissertation]. , (2016).</li><li>Seo, M. D., Enomoto, M., Ishiyama, N., Stathopulos, P. B., Ikura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+endoplasmic+reticulum+stored+calcium+regulation+by+inositol+1,4,5-trisphosphate+and+ryanodine+receptors.">Structural insights into endoplasmic reticulum stored calcium regulation by inositol 1,4,5-trisphosphate and ryanodine receptors.</a> Biochim. Biophys. Acta-Mol. Cell Res. 1853 (9), 1980-1991 (2015).</li><li>Lambert, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Calcium signaling protocols. , (1999).</li><li>Stephens, D. J., Allan, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+microscopy+techniques+for+live+cell+imaging.">Light microscopy techniques for live cell imaging.</a> Science. 300 (5616), 82-86 (2003).</li><li>Bootman, M. D., Rietdorf, K., Collins, T., Walker, S., Sanderson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+-sensitive+fluorescent+dyes+and+intracellular+Ca2++imaging.">Ca2+-sensitive fluorescent dyes and intracellular Ca2+ imaging.</a> Cold Spring Harb. 2013 (2), 83-99 (2013).</li></ol>

Live single-cell imaging of fluorescent probes, such as CatchER+, is an effective technique to analyze intricate ER/SR Ca2+ signaling processes in each cell in response to receptor agonists or antagonists. This technique is also useful for imaging using multiple wavelengths concurrently, such as needed for Fura-2 or to image CatchER+ and Rhod-2 together to monitor both ER and cytosolic calcium changes, respectively. There are several critical steps in this protocol; cell transfection can ...

The spatio-temporal attributes of intracellular calcium (Ca2+) transients activate various biological functions1. These Ca2+ signaling events are triggered extracellularly through different stimuli and controlled intracellularly by the major Ca2+ storage organelle and by numerous Ca2+ pumps, channels, and Ca2+ binding proteins. Ca2+ transients can be significantly altered as a result of defects with signal modulation, leading to different diseases2. Because of the speed and intricacy of the Ca2+ signaling system, with the endo- (ER) and sarcoplasmic reticulum (SR) at the center, genetically-encoded Ca2+ probes that have been optimized for mammalian expression with fast kinetics are needed to observe global and local Ca2+ changes in different cells3.
The ER and the SR, its counterpart in muscle cells, are the major intracellular Ca2+ storage organelles and act as Ca2+ sinks that help to amplify the Ca2+ signal4. The ER/SR is an integral part in Ca2+ signaling with dual roles as a transmitter and receiver of signals5. The ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) are Ca2+ release receptors located on the membranes of the ER/ SR that are regulated by Ca2+ 6. Other agents directly or indirectly stimulate the function of these receptors. 4-chloro-m-cresol (4-cmc) is a potent agonist of the RyR, having a 10 fold higher sensitivity than caffeine for inducing SR Ca2+ release where both are regularly employed to study RyR-mediated Ca2+ release in healthy and diseased cells7. ATP increases IP3-mediated Ca2+ release through the IP3R8. ATP binds to the purinergic receptor P2YR, a G-protein coupled receptor (GPCR), triggering the production of IP3 that binds to the IP3R to release Ca2+ from the ER9,10. The sarco-endoplasmic reticulum calcium ATPase (SERCA) pump is a P-type ATPase pump, also located on the ER/SR membrane that reduces cytosolic Ca2+ and refills the ER/SR by actively pumping the ion into the ER/SR lumen11. Specific inhibitors of the SERCA pump include thapsigargin, from Thapsia garganica, and cyclopiazonic acid (CPA), from Aspergillus and Penicillium. CPA has a low affinity for the pump and reversibly blocks the Ca2+ access point12. Thapsigargin, on the other hand, irreversibly binds to the Ca2+ free pump at residue F256 in the M3 helix with nanomolar affinity11. Analyzing and quantifying the changes involved in Ca2+ stimulated events has been and remains a challenge. Since the ER/SR is the major subcellular Ca2+ containing compartment with a central function in the propagation of the Ca2+ signal, much work has been focused on understanding ER/SR Ca2+ signaling5.
The creation of synthetic Ca2+ dyes helped to advance the field and practice of Ca2+ imaging. Although dyes, such as Mag-Fura-2, have been widely used to measure compartmentalized Ca2+ in different cells,13,14,15 they have limitations such as uneven dye loading, photobleaching, and the inability to be targeted to specific organelles. The discovery of the green fluorescent protein (GFP) and the advancement of fluorescent protein-based Ca2+ probes has propelled the field of Ca2+ imaging forward16. Some of the existing GECIs are F&#246;rster resonance energy transfer (FRET) pairs involving yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), calmodulin and the M13 binding peptide17,18. Troponin C-based GECIs are also available as FRET pairs of CFP and Citrine and as single fluorophore probes19,20,21. Others, such as GCaMP2 and R-GECO are single fluorophore sensors involving calmodulin22,23. To overcome the limitations of narrow tuning of Kd&#39;s and cooperative binding associated with multiple Ca2+ binding sites found in their Ca2+ binding domains24, a novel class of calcium sensors was created by designing a Ca2+ binding site on the surface of the beta barrel in a chromophore sensitive location of enhanced green fluorescent protein (EGFP)25,26. This highly touted sensor, called CatchER, has a Kd of ~0.18 mM, a kon near the diffusion limit, and a koff of 700 s-1. CatchER has been used to monitor receptor-mediated ER/SR calcium release in different mammalian cell lines such as HeLa, HEK293, and C2C1225. Because of its fast kinetics, CatchER was used in flexor digitorum brevis (FDB) muscle fibers of young and old Friend Virus B NIH Jackson (FVB) mice to reveal that more Ca2+ remains in the SR after 2 s of depolarization in the FDB fibers of old mice compared to that of young mice27. To overcome its low fluorescence at 37 &#176;C, which hinders its applications in calcium imaging of mammalian cells, we have developed an improved version of CatchER called CatchER+. CatchER+ exhibits enhanced fluorescence at 37 &#176;C for better application in mammalian cells. Additional mutations were incorporated into CatchER to improve the thermostability and fluorescence at 37 &#176;C28,29, to create CatchER+. CatchER+ exhibits a six-fold increase in its signal to noise ratio (SNR) over CatchER30.
Here, the protocols for the culture and transfection of HEK293 and C2C12 cells with CatchER+ and its application for monitoring ER/SR receptor-mediated calcium transients are presented. Representative results are shown for CatchER+ expressed in HEK293 cells treated with 4-cmc, CPA and ATP. We also provide a protocol for determining the in situ Kd of CatchER+ in C2C12 myoblast cells and quantification of basal [Ca2+].

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-Chloro-3-methylphenol (4-CmC)</td>
			<td>Sigma-Aldrich</td>
			<td>C55402</td>
		</tr>
		<tr>
			<td>515DCXR dichroic mirror</td>
			<td>Chroma Technology Corp.</td>
			<td>NC338059</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A26209</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>EMD Millipore</td>
			<td>102382</td>
		</tr>
		<tr>
			<td>Corning tissue-culture treated culture dishes (100 mm)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430167</td>
		</tr>
		<tr>
			<td>Corning tissue-culture treated culture dishes (60 mm)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430166</td>
		</tr>
		<tr>
			<td>Cyclopiazonic Acid (CPA)</td>
			<td>EMD Millipore</td>
			<td>239805</td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>ACROS Organics</td>
			<td>41095-0010</td>
		</tr>
		<tr>
			<td>Dow Corning 111 Valve Lubricant &#38; Sealant</td>
			<td>Warner Instruments</td>
			<td>64-0275</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D7777</td>
		</tr>
		<tr>
			<td>Ethylenebis(oxyethylenenitrilo) 
			tetraacetic Acid (EGTA)</td>
			<td>ACROS Organics</td>
			<td>409911000&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFisher</td>
			<td>26140087</td>
		</tr>
		<tr>
			<td>Fisherbrand Cover Glasses 22x40 mm</td>
			<td>Fisher Scientific</td>
			<td>&#160;12-544B</td>
		</tr>
		<tr>
			<td>Hanks&#8217; Balanced Salts (HBSS)</td>
			<td>Sigma-Aldrich</td>
			<td>H4891</td>
		</tr>
		<tr>
			<td>HEPES, Free Acid, Molecular Biology Grade</td>
			<td>EMD Millipore</td>
			<td>391340</td>
		</tr>
		<tr>
			<td>Immersion Oil without autofluorescence</td>
			<td>Leica</td>
			<td>11513859</td>
		</tr>
		<tr>
			<td>Ionomycin, Free Acid</td>
			<td>Fisher Scientific</td>
			<td>50-230-5804</td>
		</tr>
		<tr>
			<td>Leica DM6100B inverted microscope with a cooled EM-CCD camera</td>
			<td>Hamamatsu</td>
			<td>C9100-13</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>ThermoFisher</td>
			<td>11668019</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>ThermoFisher</td>
			<td>L3000015</td>
		</tr>
		<tr>
			<td>Low Profile Open Diamond Bath Imaging Chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26GLP</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate</td>
			<td>Fisher Scientific</td>
			<td>M33-500</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>ThermoFisher</td>
			<td>51985034</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>EMD Millipore</td>
			<td>PX1405</td>
		</tr>
		<tr>
			<td>Potassium Phosphate, Dibasic</td>
			<td>EMD Millipore</td>
			<td>PX1570</td>
		</tr>
		<tr>
			<td>Potassium Phosphate, Monobasic</td>
			<td>EMD Millipore</td>
			<td>PX1565</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>47036</td>
		</tr>
		<tr>
			<td>SimplePCI Image Analysis Software</td>
			<td>Hamamatsu</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher Scientific</td>
			<td>S233-3</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
		</tr>
		<tr>
			<td>Sterivex-GV 0.22 &#181;m filter</td>
			<td>EMD Millipore</td>
			<td>SVGVB1010</td>
		</tr>
		<tr>
			<td>Till Polychrome V Xenon lamp</td>
			<td>Till Photonics</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Trypsin (2.5%), no phenol red (10X)</td>
			<td>ThermoFisher</td>
			<td>15090046</td>
		</tr>
	</tbody>
</table>

monitoring er/sr calcium release with the targeted ca2+ sensor catcher+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

This section will illustrate the results that were achieved using the previously described methods using the optimized ER/SR-targeted GECI CatchER+ to monitor changes in ER/SR Ca2+ through different receptor mediated pathways.
Figure 1 illustrates ER emptying through the RyR stimulated with 200 &#181;M 4-cmc. 4-cmc is an agonist of the RyR. Addition of the drug induces a decrea...

Watch this Scientific Journal Video about Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+ at JoVE.com

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...

Research

JoVE Journal

Biology

Monitoring Actin Disassembly with Time-lapse Microscopy

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...