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) in deionized water and add 10 mM HEPES, 5 mM NaHCO3, and 1 mM EGTA. Adjust to pH 7.2-7.3 with NaOH. Filter buffer with a 0.22 &#181;m filter into an autoclaved bottle.</li>
	<li>Prepare 900 mL of high glucose Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) h in deionized water. Add 44 mM NaHCO3 and adjust the pH to 7.2-7.3 with HCl. Filter media with a 0.22 &#181;m filter into an autoclaved bottle. Add 100 mL of fetal bovine serum (FBS) to the media to make the FBS concentration 10%. Store at 4 &#176;C.</li>
	<li>Prepare 1 L of intracellular buffer (125 mM KCl, 25 mM NaCl, 10 mM HEPES, 0.2 mM CaCl2, 0.5 mM EGTA, 0.2 mM MgCl2) in deionized water, and adjust the pH to 7.25. The final [Ca2+] will be ~100 nM. Before use, add 0.5 mM ATP. Store at room temperature.</li>
	<li>Prepare 1 L of Ringer&#39;s buffer (121 mM NaCl, 2.4 mM K2HPO4, 0.4 mM KH2PO4, 10 mM HEPES, and 1.2 mM MgCl2) in deionized water, and adjust the pH to 7.2. Store at room temperature. To use, aliquot 50 mL into a 50 mL tube and add 1.8 mM Ca2+ and 10 mM glucose.</li>
	<li>Prepare 1 L of KCl rinse solution (125 mM KCl, 25 mM NaCl, 10 mM HEPES, 0.2 mM MgCl2) in deionized water, and adjust the pH to 7.25.</li>
	<li>Dissolve 5 mg of ionomycin in 1 mL of dimethyl sulfoxide (DMSO). Aliquot 30 &#181;L into microtubes and store at -20 &#176;C.</li>
	<li>Prepare the cyclopiazonic acid (CPA) stock by dissolving CPA into DMSO. Make sure the final percent of DMSO is &#8804; 1% for the CPA added to the cells to prevent apoptosis. Prepare 20 mM 4-chloro-m-cresol (4-cmc) by dissolving into filtered H2O and letting it dissolve overnight by shaking at 37 &#176;C. Prepare the ATP stock by dissolving in filtered H2O to 100 mM.</li>
	<li>To determine the in situ Kd, prepare 15 mL each of 1 mM EGTA, 0.2 mM, 0.6 mM, 2 mM, 5 mM, 10 mM, 20 mM, 50 mM, and 100 mM Ca2+ in KCl buffer using a 100 mM EGTA stock and a 1 M CaCl2 stock dissolved in deionized water.</li>
</ol>
3. Cell Culture
<ol>
	<li>Culture C2C12 myoblast and HEK293 cells according to ATCC protocols for each using 10 cm cell culture dishes in a sterile UV hood. 
	NOTE: C2C12 cells should not be allowed to grow higher than ~70% confluence since the myoblasts will begin to differentiate into myotubes. Both HEK293 and C2C12 cells grow readily and should not be allowed to grow past 100% confluency to maintain cell integrity. Additionally, cells should not be passaged more than 10 times before using them for an experiment to preserve cell health.</li>
</ol>
4. Transfection of HEK293 Cells
<ol>
	<li>Seed HEK293 cells onto sterilized 22 mm x 40 mm glass microscope slides in 6 cm dishes so they are ~70% confluent the day of transfection.</li>
	<li>The next day, follow the manufacturer&#39;s protocol for the transfection reagent to transfect cells using 2 &#181;g of CatchER+ cDNA and a 1:3 (weight/volume) DNA:transfection reagent ratio in the reduced serum media (e.g. Opti-MEM).</li>
	<li>Empty the DMEM media from the dish, and add 3 mL of reduced serum media. Add DNA:transfection reagent mixture to the dish and incubate for 4-6 h at 37 &#176;C.</li>
	<li>After incubation, wash the cells with 5-6 mL of HBSS. Discard the HBSS from the dish and replace with 3 mL of fresh DMEM and incubate them at 37 &#176;C for 48 h to allow expression of the GECI.</li>
	<li>Transfection of C2C12 myoblast cells
	<ol>
		<li>For C2C12 myoblast cells, trypsinize cells by adding enough trypsin to cover the bottom of dish (1-2 mL). Place the dish in a 37 &#176;C incubator for 2-6 min to allow the trypsin to loosen the cells from the bottom of the dish.</li>
		<li>Once the incubation is complete, remove the trypsin and aspirate the cells in 8 mL of DMEM. Pipette the cells thoroughly with the DMEM to evenly disperse and re-suspend te cells and seed them onto sterile coverslips in 6 cm cell culture dishes containing 3 mL of DMEM so that the final confluence is 60%.</li>
		<li>Transfect the cells the same day as outlined in step 4.2-4.3. Incubate for 24 h at 37 &#176;C.</li>
		<li>Repeat step 4.4.</li>
	</ol>
	</li>
</ol>
5. Preparation of Slide and Fluorescence Microscope
<ol>
	<li>Turn on microscope and light source, then open the Simple PCI program. Allow the microscope to warm up for 15-20 min or until the charge-coupled device (CCD) camera cools to -65 &#176;C. Prepare the chamber and slide with cells while waiting.</li>
	<li>Cover the bottom of the low profile open diamond bath imaging chamber with a thin layer of sealant, using a cotton swab.</li>
	<li>Take the slide containing transfected cells out of the incubator and gently rinse three times with 1 mL of Ringer&#39;s buffer with 10 mM glucose and 1.8 mM Ca2+ preheated to 37 &#176;C.</li>
	<li>Leave 1 mL of Ringer&#39;s buffer in the dish, to prevent cells from drying, and use forceps to take the slide out of the dish. Allow excess solution to absorb onto a laboratory tissue by touching the edge of the slide to a laboratory tissue. Then place onto the side of the chamber containing the grease with cells facing up towards grease and secure chamber onto the stage mount using a screwdriver.</li>
	<li>Add 1 mL of Ringer&#39;s buffer to the chamber to prevent cells from drying while mounting the chamber onto the stage and completing the rest of the microscope setup. Once the vacuum suction hose is attached to the chamber, the final volume the chamber holds is ~450 &#181;L.</li>
	<li>Switch the microscope objective to the 40X oil immersion objective.</li>
	<li>Use lens paper and lens cleaning solution to clean and dry the objective. Do not rub the objective. Dab gently until the surface is clean.</li>
	<li>Add one drop of the immersion oil to the objective.</li>
	<li>Place the mount onto the microscope stage and add the vacuum tip to the chamber. Once the vacuum suction hose is attached to the chamber and turned on, the final volume the chamber holds is ~450 &#181;L. This final volume is level with the sides of the chamber. It is imperative for the chamber solution to be level, during the experiment, to prevent the solution from spilling down into the objective due to overfilling.</li>
	<li>Raise the objective and use the coarse adjustment until the oil on the objective touches the bottom of the slide.</li>
	<li>Using the bright field mode, adjust the gain to 175 and the exposure time to 0.03 s to focus the cells.</li>
	<li>Once cells are focused, switch to fluorescence mode using 488 nm excitation. Change the exposure time to 0.07 s. Locate a field of view that has enough cells that are healthy with adequate fluorescence to image.</li>
	<li>Once a field of view has been chosen, take pictures in fluorescence and bright field mode.</li>
	<li>Circle a region of interest (ROI) in the cells or circle the whole cell to record the intensity from the chosen area.</li>
</ol>
6. Imaging Drug-induced Ca2+ Transients and In Situ Kd Calibration
<ol>
	<li>Start intensity measurement with frame rate set at one every 5 s.</li>
	<li>Allow the baseline intensity to stabilize for 20-30 frames (100-150 s). If needed, decrease the frames/s during this step to prevent photobleaching.</li>
	<li>Calculate the amount of 4-cmc needed from a 20 mM stock based on the final chamber volume of ~450 &#181;L to make a final concentration of 200 &#181;M. Make dilutions of the stock as necessary.</li>
	<li>Carefully take 60 &#181;L of Ringer&#39;s buffer from the chamber and place in a microcentrifuge tube. Dilute the calculated amount of 4-cmc with this solution and add back to the chamber to evoke the intended response from the cells being imaged.</li>
	<li>Add the calculated amount of 4-cmc to the microcentrifuge tube and mix by pipetting.</li>
	<li>Gently add the solution over the field of view while simultaneously adding the event marker in the intensity measurement.</li>
	<li>Allow time for the drug to bind to the receptor and subsequent signal decrease, then wash with 3-6 mL of Ringer&#39;s buffer with 1.8 mM Ca2+ and 10 mM glucose while simultaneously adding the event marker. Since the chamber volume is 450 &#181;L and is connected to a vacuum, adding 3-6 mL of buffer ensures that the former solution containing the drug has been washed away and replaced with the newly added solution.</li>
	<li>Repeat 6.1-6.7 for 100 &#181;M ATP and 15 &#181;M CPA, using a new slide of transfected cells for each assay.</li>
	<li>Once the measurement is complete, end the data collection in the intensity measurement window in the Simple PCI program.</li>
	<li>Take fluorescence and brightfield pictures of the cells.</li>
	<li>Open the data file for the experiment. Here, re-circle the cells or regions of interest to re-calculate the intensity values, if needed.</li>
	<li>To determine the in situ Kd in C2C12 myoblast cells, follow the protocol as outlined in section 4.2 and section 5 and put 1 mL of 0 mM Ca2+ Ringer&#39;s buffer in the chamber.
	<ol>
		<li>Permeabilize the cells with 0.002-0.005% saponin in intracellular buffer for 15-30 s to empty the cells of any CatchER+ that may be in the cytosol. Wash the cells with 3-6 mL of 1 mL EGTA in KCl buffer with 10 &#181;M ionomycin. Allow the intensity to decrease until a plateau is reached.</li>
		<li>Rinse with 3-6 mL of KCl buffer with 10 &#181;M ionomycin and allow the intensity to stabilize, then add each Ca2+ solution detailed in step 2.7. Allow the intensity to reach a plateau between each addition.</li>
	</ol>
	</li>
	<li>To calibrate the sensor for basal [Ca2+] quantification, follow step 6.12. Use the EGTA and the 100 mM Ca2+ buffer from step 2.7 to get the minimum and maximum fluorescence intensity.</li>
</ol>
7. Data Processing
<ol>
	<li>Download the spreadsheet file of the intensity measurement data.</li>
	<li>Normalize the data for each cell to the baseline intensity (F/F0). Plot the normalized data versus time in any graphing program.</li>
	<li>To calculate the % change, subtract the normalized peak value from 1 and multiply by 100. Calculate the average and standard deviation if multiple cells were imaged.</li>
	<li>To calculate the signal to noise ratio, SNR, open up the image file saved from the experiment in an image processing software, such as ImageJ, and then measure the signal, transfected cells, and noise, background. Calculate the SNR using the equation SNR = signal/noise.</li>
	<li>To calculate the in situ Kd from the collected fluorescence imaging data, average the intensity data, comprising the plateaus, after the addition of each Ca2+ concentration and EGTA (0 mM Ca2+). Calculate the fractional saturation (relative intensity) using &#952; = (F &#8211; Fmin)/(Fmax- Fmin), where &#952; is the relative intensity, F is the fluorescence at any point, Fmin is the minimum fluorescence intensity, and Fmax is the maximum fluorescence intensity, to see the change in the intensity of the probe with the addition of Ca2+ compared to the minimum intensity. Plot the relative intensity data against the [Ca2+] in any graphing and curve fitting program. Use the 1:1 binding equation &#952; = [MT]/(Kd + [MT]) translated for any graphing and curve fitting program to calculate the Kd. MT is the total metal concentration.</li>
	<li>To quantify the basal calcium from the calibration outlined in 6.13, average the intensity data, comprising the plateaus, after the addition of 1 mM EGTA (Fmin) and 100 mM Ca2+ (Fmax). Use the calibration equation [Ca2+] = Kd([F &#8211; Fmin)/(Fmax &#8211; F)] to calculate the basal calcium.</li>
</ol>

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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>

The authors have nothing to disclose.

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 have a great effect on the viability of imaging. Low transfection rates can result in insufficient expression of the GECI for monitoring calcium responses. On the other hand, overexpression of CatchER+ will not have a buffering effect on ER/ER Ca2+, a problem associated with synthetic calcium dyes. Buffering effects of Ca2+ probes are influenced by the [Ca2+] in the organelle, the Kd of the probe and the concentration of the probe required for measurement. Cytosolic [Ca2+] is typically in the low nanomolar range, however, the required probe concentration is in a comparable range. In this case, the amount of dye concentration and its capability in buffering cytosolic calcium was, and is, a major concern for cellular imaging. In contrast, ER/SR [Ca2+] is in the 100s of micromolar to millimolar range31 as we determined for different mammalian cell lines25. Since 0.1-1 &#181;M expression of CatchER+ is sufficient to enable detection of ER calcium, the buffering effect of CatchER is negligible. Thus, CatchER+ can monitor Ca2+ flux in high [Ca2+] environments without any buffering effect on luminal ER/SR Ca2+32.
Several factors can affect transfection efficiency of the GECI such as the incubation time, the transfection reagent, the incubation temperature, and the cell confluency. The cell confluency, when seeding the cells on the slide, can significantly affect the imaging quality. Low confluency can result in a low number of cells (n) and less statistical accuracy, while high levels of confluency lead to overlapping layers of cells producing large variables in imaging. The time and temperature for transfection should be optimized based on the cell type. Additionally, adding DMEM with reduced serum media is optimal for longer transfection times to prevent apoptosis. The original GECI CatchER fluorescence in mammalian cells, when cultured and transfected, was only 30 &#176;C. The fluorescence of CatchER was successfully optimized and improved for expression at 37 &#176;C resulting in the new, improved variant CatchER+. A western blot using an antibody against EGFP can be performed to confirm the expression of Ca2+ probe.
Moreover, the method and consistency of reagent delivery is critical. Reagents can be added to the chamber by perfusion, small volume diffusion, or by mechanical pumps, all of which can elicit differing responses. It is imperative that the solution chamber is properly sealed by applying a layer of sealant grease on the bottom of the chamber that will be placed onto the slide. The correct wavelengths must be selected for the probe. Excitation and emission wavelengths for CatchER+ are 395/488 nm and 510 nm, respectively. For cell imaging, only 488 nm is used for excitation to avoid the detrimental effect of UV light on cells. Therefore, using any optical filters which excite at 488 nm and collect the emission at 510 nm would be acceptable to use to image with CatchER+. To prevent photobleaching, efforts may be focused to optimize light intensity and light exposure such as frames/s33.
While this protocol is ideal to analyze the effect of ER/SR changes using CatchER+ Ca2+ ER/SR fluorescent probe, there are limitations as expected in any experiment. Since single-cell live imaging only analyzes a small frame of cells, the number of cells (n) can vary from 1-100 cells, on average, but can be lower for larger cell lines. This leads to lower cell numbers and statistically varied results without multiple trials. An n&#62;6 is required for statistical accuracy. To get a larger n, many dishes must be imaged or other techniques must be used, concurrently. Additionally, CatchER+ is a single wavelength ER/SR Ca2+ probe, this leads to the issues of other single wavelength probes and dyes with not being able to quantify the signal, not knowing if signal is true as opposed to disruption of the cells artifact, and having larger noise as compared to ratiometric systems34.
This work shows that these optimized Ca2+ probes can be applied in different cell types or tissue types to monitor receptor-mediated ER/SR Ca2+ release. CatchER+ is a single wavelength ER/SR Ca2+ probe. Therefore, it is important that the experimental parameters and settings are consistent for quantitative measurement. A detailed calibration of calcium concentration and Kd of calcium sensor in the cell lines are additional important measurements for quantitative analysis.
These developed calcium sensors can also be targeted to specific locations within the ER/SR to monitor the vastly different Ca2+ transients that exist compared to global Ca2+ changes in various biological and pathological conditions. These findings will continue to push the fields of Ca2+ imaging and probe design forward to provide future tools for diagnosing Ca2+-related diseases. The reported protocols and developed sensor can also be adapted for drug discoveries against diseases associated with calcium signaling.

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><tbody><tr><td>4-Chloro-3-methylphenol (4-CmC)</td><td>Sigma-Aldrich</td><td>C55402</td><td></td></tr><tr><td>515DCXR dichroic mirror</td><td>Chroma Technology Corp.</td><td>NC338059</td><td></td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate disodium salt hydrate</td><td>Sigma-Aldrich</td><td>A26209</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>EMD Millipore</td><td>102382</td><td></td></tr><tr><td>Corning tissue-culture treated culture dishes (100 mm)</td><td>Sigma-Aldrich</td><td>CLS430167</td><td></td></tr><tr><td>Corning tissue-culture treated culture dishes (60 mm)</td><td>Sigma-Aldrich</td><td>CLS430166</td><td></td></tr><tr><td>Cyclopiazonic Acid (CPA)</td><td>EMD Millipore</td><td>239805</td><td></td></tr><tr><td>D(+)-Glucose</td><td>ACROS Organics</td><td>41095-0010</td><td></td></tr><tr><td>Dow Corning 111 Valve Lubricant &amp;amp; Sealant</td><td>Warner Instruments</td><td>64-0275</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Sigma-Aldrich</td><td>D7777</td><td></td></tr><tr><td>Ethylenebis(oxyethylenenitrilo)&lt;br/&gt; tetraacetic Acid (EGTA)</td><td>ACROS Organics</td><td>409911000&amp;nbsp;</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher</td><td>26140087</td><td></td></tr><tr><td>Fisherbrand Cover Glasses 22x40 mm</td><td>Fisher Scientific</td><td>&amp;nbsp;12-544B</td><td></td></tr><tr><td>Hanks&amp;rsquo; Balanced Salts (HBSS)</td><td>Sigma-Aldrich</td><td>H4891</td><td></td></tr><tr><td>HEPES, Free Acid, Molecular Biology Grade</td><td>EMD Millipore</td><td>391340</td><td></td></tr><tr><td>Immersion Oil without autofluorescence</td><td>Leica</td><td>11513859</td><td></td></tr><tr><td>Ionomycin, Free Acid</td><td>Fisher Scientific</td><td>50-230-5804</td><td></td></tr><tr><td>Leica DM6100B inverted microscope with a cooled EM-CCD camera</td><td>Hamamatsu</td><td>C9100-13</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>ThermoFisher</td><td>11668019</td><td></td></tr><tr><td>Lipofectamine 3000 Transfection Reagent</td><td>ThermoFisher</td><td>L3000015</td><td></td></tr><tr><td>Low Profile Open Diamond Bath Imaging Chamber</td><td>Warner Instruments</td><td>RC-26GLP</td><td></td></tr><tr><td>Magnesium Chloride Hexahydrate</td><td>Fisher Scientific</td><td>M33-500</td><td></td></tr><tr><td>Opti-MEM</td><td>ThermoFisher</td><td>51985034</td><td></td></tr><tr><td>Potassium Chloride</td><td>EMD Millipore</td><td>PX1405</td><td></td></tr><tr><td>Potassium Phosphate, Dibasic</td><td>EMD Millipore</td><td>PX1570</td><td></td></tr><tr><td>Potassium Phosphate, Monobasic</td><td>EMD Millipore</td><td>PX1565</td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>SimplePCI Image Analysis Software</td><td>Hamamatsu</td><td>N/A</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Fisher Scientific</td><td>S233-3</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>S271-500</td><td></td></tr><tr><td>Sterivex-GV 0.22 &amp;micro;m filter</td><td>EMD Millipore</td><td>SVGVB1010</td><td></td></tr><tr><td>Till Polychrome V Xenon lamp</td><td>Till Photonics</td><td>N/A</td><td></td></tr><tr><td>Trypsin (2.5%), no phenol red (10x)</td><td>ThermoFisher</td><td>15090046</td><td></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 decrease in ER calcium as measured by the decrease in the CatchER+ fluorescence intensity. As marked, the protocol outlined here allows the visualization and measurement of receptor-mediated Ca2+ transients using CatchER+ which provides information about the changes in ER Ca2+.
As depicted in Figure 2, the addition of 15 &#181;M CPA, to reversibly block the SERCA pump, produces a large decrease in the fluorescence intensity that forms a plateau. As the function of the SERCA pump is inhibited, the ER Ca2+ release channels are still actively releasing Ca2+ into the cytosol causing the fluorescence intensity to decrease. The cells recover after washing CPA away with Ringer&#39;s buffer containing 1.8 mM Ca2+ and 10 mM glucose. These results confirm the ability of CatchER+ to monitor Ca2+ transients specific to the SERCA pump.
Figure 3 shows representative data for the indirect activation of the IP3R with 200 &#181;M ATP in HEK293 cells transfected with CatchER+. ATP binds to the P2Y receptor, a G-protein coupled receptor, which generates IP3 that activates Ca2+ release through the IP3R. Although the change is small, the addition of 200 &#181;M ATP produces a visible decrease in signal intensity. Washing the cells with Ringer&#39;s buffer containing 1.8 mM Ca2+ and 10 mM glucose allows the ER to refill and the signal to return to the baseline. These results highlight the ability of CatchER+ to monitor IP3R-mediated calcium release through indirect stimulation.
In Figure 4, CatchER+ was transfected into C2C12 myoblast cells to determine the in situ Kd. The data was collected as outlined in the protocol. C2C12 myoblast cells were permeabilized with 0.005% saponin for 15-30 s then washed with 1 mM EGTA in KCl buffer containing 10 &#181;M ionomycin. The various Ca2+ concentrations were prepared in KCl buffer containing 10 &#181;M ionomycin and were added in a stepwise manner. The data was normalized and fitted using the 1:1 binding equation. The average Kd was 3.1 &#177; 1.4 mM for 7 cells. The weak affinity of CatchER+ allows it to sense Ca2+ in high Ca2+ organelles like the ER or SR. To determine the basal [Ca2+] in the SR, C2C12 cells were permeabilized with 0.005% saponin for 15-30 s, washed with KCl buffer, and then washed with 1 mM EGTA in KCl buffer containing 10 &#181;M ionomycin to obtain Fmin. After washing the EGTA away with KCl buffer a maximum, Fmax, of 100 mM Ca2+ with 10 &#181;M ionomycin was added. The basal Ca2+ was calculated to be 0.4 &#177; 0.2 mM.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55822/55822fig1.jpg" /> 
Figure 1: Stimulation of RyR using 4-cmc in HEK293 cells. (A) Depiction of the activation of RyR with 4-cmc. (B) Intensity recording of HEK293 cells transfected with CatchER+, localized in the ER, treated with 200 &#181;M 4-cmc. Stimulation of the RyR with 4-cmc causes a decrease in the normalized fluorescence intensity that directly correlates to a decrease in [Ca2+]ER. n refers to the number of cells imaged. The average signal decrease was 12.0 &#177; 2.2%. The presence of 1.8 mM Ca2+ in the Ringer&#39;s buffer facilitated refilling of the ER reflected in the intensity recovery to baseline. Inset pictures show the cells before and after treatment with 4-cmc. SNR was calculated to be 4.3 &#177; 0.8. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55822/55822fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55822/55822fig2.jpg" /> 
Figure 2: Decrease in [Ca2+]ER using the reversible SERCA pump inhibitor CPA in HEK293 cells. (A) The inhibition of the SERCA pump by CPA. (B) Live imaging of HEK293 cells transfected with CatchER+, localized in the ER, treated with 15 &#181;M CPA. Because Ca2+ can still flow through the IP3R and RyR, blocking the SERCA pump with CPA causes a decrease in the normalized fluorescence intensity that directly correlates to a decrease in [Ca2+]ER. n refers to the number of cells imaged. Inset pictures are HEK293 cells transfected with CatchER+ before and after treatment. The average signal decrease was 21.0 &#177; 0.3%. The presence of 1.8 mM Ca2+ in the Ringer&#39;s buffer facilitated refilling of the ER reflected in the intensity recovery to baseline. SNR was calculated to be 5.5 &#177; 0.8. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55822/55822fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55822/55822fig3.jpg" /> 
Figure 3: ATP decreases [Ca2+]ER in HEK293 cells. (A) Schematic of the indirect activation of IP3R with ATP through the purinergic receptor P2YR. P2YR is a GPCR that generates IP3 upon ATP binding. The IP3 activates the IP3R, stimulating calcium release from the ER. (B) Real time imaging of HEK293 cells transfected with CatchER+, localized in the ER, treated with 200 &#181;M ATP. Indirect stimulation of the IP3R via the P2Y receptor with ATP causes a decrease in the normalized fluorescence intensity that directly correlates to a decrease in [Ca2+]ER. n refers to the number of cells imaged. The average signal decrease was 6.0 &#177; 3.0%. SNR was calculated to be 6.7 &#177; 2.7. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55822/55822fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55822/55822fig4.jpg" /> 
Figure 4: In situ Kd of CatchER+ in C2C12 myoblast cells. Representative trace for normalized intensity collected from permeabilized C2C12 myoblast cells transfected with CatchER+. Cells were permeabilized with 0.005% saponin in intracellular buffer for 15-30 s. EGTA and Ca2+ solutions were prepared in KCl buffer, n refers to the number of cells imaged. (A) Inset fluorescence images are representative of cells before and after treatment. 0.3, 0.6, 2, 5, 10, 20, 50, and 100 mM Ca2+ was added to permeabilized C2C12 myoblast cells in the presence of 10 &#181;M ionomycin to get a Kd of 3.1 &#177; 1.4 mM. (B) Inset fluorescence images are representative of cells at minimum and maximum values, after addition of 1 mM EGTA and 100 mM Ca2+ with 10 &#181;M ionomycin each, respectively. The basal Ca2+ was calculated to be 0.4 &#177; 0.2 mM. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55822/55822fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

monitoring-ersr-calcium-release-with-the-targeted-ca2-sensor-catcher

Universidad San Sebastian

Research

JoVE Journal

Biology

14.8K Views.  Georgia State University. 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.

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

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

Neuroscience

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

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 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

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

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

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

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

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 Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

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

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

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

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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 levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

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

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.

The present protocol describes all-optical control and observation of triggered cellular activity in iPSC-derived cardiomyocytes (iPSC-CMs) for high throughput drug screening and toxicity testing. Multi-parametric quantification of phenotypic patterns in time, and space, are shown. Long-term effects of drugs over hours, or sequential measurements over days, are demonstrated.

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. ...

Medicine

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

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

Summary

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Chapters in this video

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

In vivo Neuronal Calcium Imaging in C. elegans

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

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

Dissection of Local Ca²⁺ Signals in Cultured Cells by Membrane-targeted Ca²⁺ Indicators

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

Imaging Local Ca²⁺ Signals in Cultured Mammalian Cells

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging