This work was supported by the Intramural Research Program at the National Institute on Drug Abuse. We thank Doug Howard, Chris Richie, Lowella Fortuno, and Josh Hinkle for their contributions to developing this method.

1. In Vitro&#160;Assay: Detecting SERCaMP Release from a Stable SH-SY5Y Cell Line
<ol>
	<li>Plate SH-SY5Y-GLuc-ASARTDL (SERCaMP) in tissue culture treated plates at 150,000&#160;cells per cm2 of surface area. For 96 well plates, for example, seed 50,000 cells per well (Figure 1A). Grow SH-SY5Y cells in DMEM (high glucose, GlutaMAX, pyruvate) + 10% bovine growth serum + 1x penicillin/streptomycin.
	<ol>
		<li>Passage cells up to 15 times (Figure 1B). Higher passage number has not been tested.</li>
		<li>Return cells to humidified incubator at 37 &#176;C with 5.5% CO2 and incubate overnight.</li>
	</ol>
	</li>
	<li>Before experimental treatment(s), collect a baseline sample for each well by transferring 5 &#181;l of culture supernatant to an opaque walled 96 well plate. Before collecting, gently tap the plate on all sides and swirl to avoid gradient effects. Seal the opaque plate with an adhesive sealing sheet and store at 4 &#176;C until time of enzymatic assay. 
	Note: Secreted GLuc-SERCaMP is very stable in cell culture medium. We previously reported approximately 5-10% of GLuc activity was lost after a 72 hr incubation at 37 &#176;C (incubated on SH-SY5Y cells)12.</li>
	<li>Treat cells as desired to examine ER calcium depletion (e.g. environmental, pharmacologic, genetic manipulations). Avoid long exposure to ambient environment (outside of the controlled incubator) as pH changes will rapidly occur at atmospheric CO2. Add HEPES to the culture medium at 20 mM, to stabilize pH14, if prolonged manipulations are required. Avoid exposure to light when using HEPES in culture medium15.
	<ol>
		<li>Positive control: Treat cells with 100 nM thapsigargin (Tg) or 50 &#181;M cyclopiazonic acid (CPA) for 4-6 hr for experiments in SH-SY5Y cells. Maximal response in other cell lines may require longer incubations or different concentrations of compounds.</li>
		<li>Negative control: Collect culture medium from parental SH-SY5Y cells. In our experience, the background luminescence for this assay is less than 0.05% of basal secretion from stable SH-SY5Y-GLuc-SERCaMP cells.</li>
	</ol>
	</li>
	<li>Collect and store 5 &#181;l samples of conditioned media at the desired timepoints, as outlined in step 1.2.</li>
	<li>Assess enzymatic activity in the medium as outlined in Section 5.</li>
	<li>Examine intracellular GLuc by immunoblot or luminescence assay.
	<ol>
		<li>For immunoblot: lyse cells in modified RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.25% sodium deoxycholate, 1 mM EDTA, 1% NP40, and protease inhibitors. Separate on SDS-polyacrylamide gel and transfer to 0.20 &#181;m PVDF membrane. Incubate membrane with GLuc antibody (1:2,000 dilution) overnight at 4 &#176;C. Perform secondary antibody incubation and membrane washes as per user-defined protocol for detection reagent of choice.</li>
		<li>For luminescence assay (Figure 2): perform the following steps on ice:
		<ol>
			<li>Remove all medium from wells of the tissue culture plate and rinse with 200 &#181;l of cold PBS.</li>
			<li>Add 75 &#181;l of lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40 and protease inhibitors to each well.</li>
			<li>Rotate the plate on an orbital shaker (120 rpm) at 4 &#176;C for 20 min.</li>
			<li>Gently pipette the lysate up and down using a multichannel pipettor, avoiding air bubbles. Transfer 5 &#181;l of lysate to an opaque plate and assess luminescence as outlined in Section 5.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. In Vitro&#160;Assay: Transient Transfection of Immortalized Cells with SERCaMP
Note: We have observed that transient transfection procedures can induce cellular stress and blunt the subsequent GLuc-SERCaMP response. The following approach was developed to minimize transfection effects in SH-SY5Y cells. Optimization of this procedure (including choice of transfection reagent) for alternate cell lines may be required. When possible, it is recommended to use stable cell lines or viral transduction methods outlined in Sections 1 and 3 respectively.
<ol>
	<li>Plate SH-SY5Y cells in a 100 mm dish at 4 x 106 cells. This dish will be sufficient to re-seed approximately 300 wells (96 well plate format) following the transfection procedure.</li>
	<li>Transfect cells with Xfect reagent using 20 &#181;g of plasmid DNA (encoding GLuc-SERCaMP) and 6 &#181;l of Xfect reagent. Scale DNA and Xfect reagent accordingly for larger or smaller culture vessels.</li>
	<li>Return cells to incubator for 48 hr.</li>
	<li>Trypsinize cells and reseed to 96 well plates at 60,000 cells per well. Follow subsequent steps outlined in Section 1.</li>
</ol>
3. In Vitro&#160;Assay: Viral Vector-mediated Expression of GLuc-SERCaMP
Note: Adeno-associated viral (AAV) vector packaging16 and purification12 and lentivirus production13 have been previously reported.
<ol>
	<li>Titer GLuc-SERCaMP AAV using the following primers and probes: forward primer, 5&#8242;-CACGCCCAAGATGAAGAAGT-3&#8242;; reverse primer, 5&#8242;-GAACCCAGGAATCTCAGGAATG-3&#8242;; probe (5&#8242;-6-FAM/3&#8242;-BHQ-1 labeled), 5&#8242;-TACGAAGGCGACAAAGAGTCCGC-3&#8217; for quantitative PCR.
	<ol>
		<li>Prepare the standard curve by linearizing a plasmid containing GLuc and purifying the DNA using a spin-column (e.g. Machery-Nagel NucleoSpin Gel and PCR cleanup). Quantitate the DNA by separating on an agarose gel alongside a mass ladder. Prepare 1:10 dilutions of DNA from 100 pg/ml to 10 fg/ml in PBS. Calculate the copies/ml of GLuc plasmid DNA at each of the concentrations based on the molecular weight of the plasmid.</li>
		<li>Dilute the AAV stocks in PBS (appropriate dilution will be dependent on parameters of viral preparation and determined empirically).</li>
		<li>Run the PCR reaction in a real-time PCR system using the following conditions: 95 &#176;C &#215; 5 min, 94 &#176;C &#215; 20 sec, and 60 &#176;C &#215; 1 min for 41 cycles. Calculate copies/mL using the standard curve.</li>
	</ol>
	</li>
	<li>Titer lentivirus with a p24 Lenti-X rapid titer kit. Thaw lentiviral aliquots on ice and dilute the p24 control protein in SH-SY5Y medium.
	<ol>
		<li>Dilute the lentivirus such that it will fall on the standard curve (e.g. 1:20,000 or 1:100,000). The dilution factor required will vary based on the lentiviral preparation parameters and must be determined empirically. Follow the manufacturer&#8217;s instructions for all subsequent steps.</li>
	</ol>
	</li>
	<li>Plate cells to 96 well plates. For SH-SY5Y cells, plating procedures outlined in Section 1 can be followed. For rat primary cortical neurons, cells should be isolated and seeded on appropriately coated plates as previously described16. Maintain rat primary cortical neurons in Neurobasal medium supplemented with 1x B27 and 500 &#181;M L-glutamine. Perform half medium exchanges every other day.&#160;</li>
	<li>Transduce the cells with virus. The goal of viral transduction is to achieve low level expression, as Gaussia luciferase provides robust signal.
	<ol>
		<li>AAV transduction SH-SY5Y cells: Transduce the day following plating. Dilute AAV to 6.0 x 107 vg/&#181;l in AAV dilution buffer (PBS + 0.5mM MgCl2). Add 5 &#181;l of diluted AAV (3.0 x 108 vg; MOI of approximately 6,000) per well of 96 well plate (Figure 3A).&#160;Use 10% bleach to inactivate viral vector waste.</li>
		<li>AAV transduction of rat primary cortical neurons: Transduce 6-8 days after plating. Dilute AAV to 4.0 x 106 vg/&#181;l in AAV dilution buffer. Add 5 &#181;l of diluted AAV (2.0 x 107 vg; MOI of approximately 350) per well of 96 well plate (Figure 3B). The optimal MOI should be determined empirically for other cell types. Use 10% bleach to inactivate viral vector waste.</li>
		<li>Lentiviral transduction of SH-SY5Y cells: Transduce the day following plating. Dilute lentivirus to 20 pg/&#181;l p24 (concentration determined using titering kit) in Hank&#8217;s balanced salt solution. Add 5&#181;l of the diluted virus per well (100 pg of p24, equivalent to 1,250,000 lentiviral particles, or ~1,250 IFUs) of a 96 well plate containing 100 &#181;l volume. Scale accordingly for larger formats. Use 10% bleach to inactivate viral waste.</li>
	</ol>
	</li>
	<li>Collect a pre-treatment sample of medium and begin experimental treatments 48 hr (SH-SY5Y) or 5-7 days (rat primary cortical neurons) after transduction.</li>
</ol>
4. In Vivo SERCaMP Assay
Note: Before conducting&#160;any animal procedures be sure to obtain proper approval through your institution. All survival surgeries are to be done under sterile conditions with adequate anesthesia. All procedures described below have been approved and are in compliance with NIH ACUC guidelines.
<ol>
	<li>Autoclave surgical instruments&#160;prior to start of surgery (121 &#176;C, 30 min sterilization, 20 min dry time). Clean all instruments&#160;in an ultrasonicator immediately following all procedures and prior to autoclaving. Maintain sterile conditions during survival surgery. For surgeries requiring multiple animals, wipe surgical instruments&#160;with 70% alcohol, ultrasonicate and bead sterilize between rats. Refer to Materials List for necessary instruments.</li>
	<li>Prepare rat for surgery. Here we use male Sprague-Dawley (180-200 g).
	<ol>
		<li>Anesthetize rats using gas isoflurane for 3 min (4-5% Isoflurane delivered at 1,000 cc/min) followed by intraperitoneal injections of xylazine (8 mg/kg) and ketamine (80 mg/kg). Apply ophthalmic ointment to protect corneas from drying out. Begin surgery once the rat is deeply anesthetized as demonstrated by lack of withdrawal reflex following tail or foot pinch.</li>
		<li>Shave the region of abdomen slightly below the ribs (low thoracic region) to the mid abdominal region. Scrub the surgical area three times, alternating 70% alcohol and Betadine scrub. Place rat in supine position on sterile surgical area with sterile surgical drapes.</li>
	</ol>
	</li>
	<li>Dilute AAV-GLuc-SERCaMP to 7.6 x 109 vg/ml (final concentration). Mix well by inverting the tube. 
	Note: Range of viral concentration can vary (Figure 4).
	<ol>
		<li>Pipet 105 &#181;l of diluted AAV into a sterile dish. Use a 30-gauge needle to collect the virus into a syringe.</li>
	</ol>
	</li>
	<li>Perform surgery to expose the liver and inject AAV-GLuc-SERCaMP.
	<ol>
		<li>Prior to making an incision in the abdomen, apply 0.25% bupivacaine to the incision area.&#160; Using a scalpel, make a horizontal incision below rib cage (approximately 2-3 cm). Blunt dissect to separate connective tissue from hypodermis.</li>
		<li>Cut abdominal muscle, exposing the right medial lobe of liver. 
		Note: additional lobes may be injected based on end application.</li>
		<li>Place animal under surgical microscope and adjust to get right medial lobe of liver in the field of view. Inject virus into parenchyma of the medial lobe; 3 separate sites, approximately 33 &#181;l per site. 
		Note: Leave needle in tissue for 5-10 sec following injection to ensure delivery of the entire injection volume.</li>
		<li>Suture the abdominal muscle and skin separately and add Neosporin using cotton tipped applicator. Place rat in a recovery chamber until consciousness is regained and rat is able to maintain upright position. Do not leave rat(s) unattended until it has regained sufficient consciousness to maintain sternal recumbency. &#160; House singly until the incision has healed and sutures have been removed (7-14 days). &#160; 
		Note: As per NIH post-surgical guidelines, maintain surgical record.&#160; Annotate cage card with procedure, date, experiment identifier, body weight, and day of surgery. Pain/distress, feces production, activity, and food and water consumption are to be monitored and recorded 3 days post- surgery. Post-operative analgesia is provided by adding acetaminophen to the drinking water (450 mg/100 cc) although we do not typically observe signs of pain or distress following surgery.</li>
	</ol>
	</li>
	<li>Begin tail blood collection 4-7 days after injection. Prepare blood collection tubes by labeling and pre-weighing. Add 50 &#181;l heparin (1,000 U/ml) to each tube.</li>
	<li>Place rat in isoflurane anesthesia chamber for 3 min (4-5% Isoflurane delivered at 1,000 cc/min). Remove rat from chamber and place in nose cone (2-3% Isoflurane delivered at 500 cc/min). Blood collection can begin once the rat is deeply anesthetized as demonstrated by lack of withdrawal reflex following tail pinch.
	<ol>
		<li>Using sterile scissors cut tip of tail (1-2 mm) and collect blood drop-wise into pre-filled heparin tube. Collect blood until volume reaches greater than 2:1 blood to heparin ratio (&#62;100 &#181;l blood/50 &#181;l heparin). Blood collection volumes can be adjusted based on experimental design. Use a wet cotton applicator to apply styptic powder to the tail to stop bleeding.</li>
		<li>Store blood tubes at 4 &#176;C if collecting subsequent samples. Wipe scissors with ethanol pad and bead sterilize between collections.</li>
		<li>Weigh collection tubes and adjust with heparin to obtain 2:1 ratio (blood:heparin). This step will normalize the amount of heparin in each sample (Table 1).</li>
		<li>Centrifuge tubes at 2,000 x g for 5 min at 4 &#176;C. Transfer the supernatant (plasma) to a fresh tube and store at -80 &#176;C until time of luciferase assay (section 5). 
		Note: Storage of samples at 4 &#176;C up to 72 hr prior to enzymatic assay has minimal effect on luminescence (Figure 5A). Up to 3 freeze-thaw cycles of plasma samples have no effect on luminescence (Figure 5B).</li>
	</ol>
	</li>
	<li>Thapsigargin administration (positive control):
	<ol>
		<li>Prepare thapsigargin by diluting in ethanol to a final concentration of 2.5 mg/ml. Inject thapsigargin at 1 mg/kg i.p. into the lower abdomen. 
		Note: Thapsigargin increases thrombin-induced platelet coagulation17 and can make blood collection from tail more difficult.</li>
	</ol>
	</li>
	<li>Gaussia luciferase assay:
	<ol>
		<li>Thaw the plasma samples on ice. 
		Note: For longitudinal studies, thaw and perform luminescence assay for all timepoint samples on the same day (using single preparation of substrate).</li>
		<li>Transfer 10 &#181;l of plasma to an opaque walled plate and measure enzymatic activity as described in section 5. Run 3-4 technical replicates of each plasma sample.</li>
	</ol>
	</li>
	<li>Euthanasia 
	Note: The advantage of SERCaMP technology is the ability to longitudinally monitor ER calcium. Depending on experimental parameters and endpoint analyses, animals are to be euthanized by measures appropriate for endpoint analyses and in accordance with institutional ACUC guidelines.</li>
</ol>
5. Luminescence Assay
<ol>
	<li>Prepare coelenterazine (CTZ) stock solutions by diluting the compound in acidified methanol (30 &#181;l of 10 N HCl to 3 ml&#160;of methanol) to 20 mM. Prepare single use aliquots and store at -80 &#176;C.</li>
	<li>Prepare the working substrate on the day of assay.
	<ol>
		<li>For in vitro assays, dilute coelenterazine to 8 &#181;M in PBS, e.g. add 10 &#181;l of 20 mM CTZ stock to 25 ml&#160;of PBS (Figure 6A, B).</li>
		<li>For in vivo assays, dilute coelenterazine to 100 &#181;M in PBS, 500 mM ascorbic acid, 5 mM NaCl.</li>
	</ol>
	</li>
	<li>Incubate prepared CTZ for at least 30 min at room temperature before beginning the assay. 
	Note: This step is often included in Gaussia luciferase assays as there are reports of rapid substrate decay during first 30 min after preparing. We have not observed this effect in our system (Figure 6C); however, we often include the incubation step as we have found no negative impact on the assay.</li>
	<li>Use a plate&#160;reader that is capable of monitoring bioluminescence and equipped with a substrate injector. Prime the lines with substrate. 
	Note: The initial substrate through the lines can be prone to degradation. To avoid artificially low readings for early samples, inject 20-30 empty wells (loading an empty plate on the reader) before measuring experimental samples.</li>
	<li>Inject 100 &#181;l of substrate into well, shake medium speed for 5 sec, and measure light emission. For the Biotek Synergy II plate reader, integrate light emission over 0.5 sec (in vitro samples) and 5 sec (in vivo samples) for the read step. Optimize plate reader parameters for ideal assay performance. 
	Note: Gaussia luciferase exhibits flash kinetics with rapid signal decay (compared to glow kinetics observed with other luciferases). The flash kinetics of GLuc is influenced by serum in cell culture medium (Figure 7A), highlighting the importance of controlling for medium composition across samples. Due to the rapid decay of luminescence after adding substrate (flash kinetics), the time between inject and read steps must be uniform for all samples. The plate reader should be set to inject substrate to a well, wait a fixed time (e.g. we use a 5 sec shake step), and read that well. Adding substrate to an entire plate before reading poses a significant challenge unless substrate can be added to all wells simultaneously. If an injector is not available, the issue can be partially circumvented by incubating the plate for 10 min between substrate addition and measurement (thus avoiding the steep part of the decay curve) (Figure 7B).</li>
	<li>Repeat the injection, wait time, and read steps for each well on the plate.</li>
</ol>

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The authors have nothing to disclose.

This protocol highlights the in vitro and in vivo utility of GLuc-SERCaMP to monitor depletion of ER calcium. Although the protein modification to generate SERCaMP appears to generalize to other reporter proteins12, we chose Gaussia luciferase for its robust (200-1,000 fold greater) bioluminescence compared to other luciferases18. We demonstrate detectable thapsigargin-induced GLuc-SERCaMP release across a 100-fold dose range of GLuc-SERCaMP virus delivered to primary rat ...

The endoplasmic reticulum (ER) functions in many cellular capacities including protein folding, protein secretion, lipid homeostasis, and intracellular signaling1. Central to normal ER function is maintaining luminal calcium concentrations at ~5,000 times those found in the cytoplasm2-4. This energy intensive process is regulated by the sarco/endoplasmic reticulum calcium ATPase (SERCA), a pump that moves calcium ions into the ER. Efflux of calcium from the ER is mediated primarily by the ryanodine (RyR) and inositol triphosphate (IP3R) receptors. Because many ER processes are dependent on calcium, disrupting the store can lead to ER stress and eventual cell death.
ER calcium dysregulation has been observed in diseases including cardiomyopathy, diabetes, Alzheimer&#8217;s, and Parkinson&#8217;s5. Owing to the progressive nature of these diseases, it has been challenging to delineate the cause-effect relationship between pathogenesis and alterations in the ER calcium store. A number of technologies have allowed for significant advances in our understanding of ER calcium dynamics, including dyes and genetically encoded calcium indicators (GECIs). Low affinity calcium dyes, which increase in fluorescence when bound to Ca2+, can be loaded into cells to examine subcellular compartments with high concentrations of calcium6. GECIs, such as D1ER and CatchER allow for monitoring of calcium fluctuations with more precise control of subcellular localization7-9. Recently, another class of GECIs called calcium-measuring organelle-entrapped protein indicators (CEPIA) have been described10. A third approach combining genetics and small molecule chemistry is targeted-esterase dye loading (TED), which utilizes a genetically encoded carboxylesterase (targeted to the ER) with an ester-based calcium dye11.
While the aforementioned approaches have inherent strengths and weaknesses, they can provide valuable insight into ER calcium dynamics through acute measurements of fluorescence. They are, however, not optimal for the longitudinal studies often required to investigate disease progression. With the goal of devising a method to monitor calcium dynamics over extended periods of time, we identified and developed a protein modification to create the secreted ER calcium monitoring proteins (SERCaMPs)12.
SERCaMP circumvents several limitations associated with other methodologies, by providing a minimally invasive approach to repeatedly interrogate the ER calcium store. We have previously demonstrated that the carboxy-terminal peptide ASARTDL (alanine-serine-alanine-arginine-threonine-aspartic acid-leucine) is sufficient to promote ER retention; however, under conditions that cause decreases in ER calcium, the peptide sequence is no longer able to retain ER localization and the protein is secreted13. The basis of the SERCaMP technology is the appendage of ASARTDL to the carboxy-terminus of a secreted protein (e.g. Gaussia luciferase, or GLuc) such that secretion is triggered by ER calcium depletion, thus creating a robust reporter of ER calcium dysregulation12. The expression of GLuc-SERCaMP via transgenic methods enables biological fluids including cell culture medium and plasma to be analyzed for changes in GLuc activity as an indicator of ER calcium homeostasis. The method has applications for the longitudinal study of progressive alterations in the ER calcium store both in vitro and in vivo. The following protocol is written as a general outline for using GLuc-based SERCaMP to study ER calcium homeostasis, but the protocol can serve as a guide for alternative reporter SERCaMPs.

<table><tbody><tr><td>1.5 ml tubes</td><td>Fisher&amp;nbsp;</td><td>02-682-550</td><td></td></tr><tr><td>10% NP-40 solution&amp;nbsp;</td><td>Pierce</td><td>28324</td><td>for intracellular GLuc assays</td></tr><tr><td>1 ml luer-lok syringes</td><td>Fisher</td><td>14-823-30</td><td></td></tr><tr><td>200 microliter filter tips</td><td>Rainin</td><td>RT-L200F</td><td></td></tr><tr><td>3-0 surgical sutures</td><td>Fisher</td><td>NC9598192</td><td></td></tr><tr><td>30 G needles</td><td>Fisher Scientific</td><td>14-821-13A&amp;nbsp;</td><td></td></tr><tr><td>Adhesive microplate sealing sheets</td><td>Thermo</td><td>AB-0558</td><td></td></tr><tr><td>Alcohol prep pads</td><td>Fisher</td><td>22-246-073</td><td></td></tr><tr><td>Anesthesia Auto Flow System</td><td>E-Z Anesthesia</td><td>EZ-AF9000</td><td></td></tr><tr><td>Animal recovery chamber</td><td>Lyon Vet</td><td>ICU-912-004</td><td></td></tr><tr><td>B27 supplement</td><td>Life Technologies</td><td>17504-044</td><td></td></tr><tr><td>Betadine solution</td><td>Fisher</td><td>NC9386574</td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td>n/a</td><td></td></tr><tr><td>Bovine growth serum</td><td>Thermo</td><td>SH30541.03</td><td></td></tr><tr><td>Coelenterazine, Native</td><td>Regis Technologies</td><td>1-361204-200</td><td></td></tr><tr><td>Cotton tipped applicators</td><td>Puritan</td><td>806-WC</td><td></td></tr><tr><td>Cutting needles 3/8 circle sutures</td><td>WPI</td><td>501803</td><td></td></tr><tr><td>Digital ultrasconic cleaner</td><td>Fisher Scientific</td><td>FS60D</td><td></td></tr><tr><td>DMEM high glucose, GlutaMAX, pyruvate</td><td>Life Technologies</td><td>10569-010</td><td></td></tr><tr><td>DNA mass ladder</td><td>Life Technologies</td><td>10496-016</td><td></td></tr><tr><td>Gaussia luciferase (recombinant protein)</td><td>Nanolight</td><td>321-100</td><td></td></tr><tr><td>Gaussia luciferase antibody (for WB, ICC, or IHC)</td><td>New England Biolabs</td><td>E8023S</td><td>1:2,000 (WB)</td></tr><tr><td>Germinator 500</td><td>CellPoint Scientific</td><td>DS-401</td><td></td></tr><tr><td>Gluc assay plates (96 well, opaque)</td><td>Fisher</td><td>07-200-589</td><td></td></tr><tr><td>Hank&#039;s balanced salt solution</td><td>Life Technologies</td><td>14175-095</td><td></td></tr><tr><td>Heparin</td><td>Allmedtech</td><td>63323-276-02</td><td></td></tr><tr><td>Isoflurane</td><td>Butler Schein</td><td>29404</td><td></td></tr><tr><td>Ketamine</td><td>Henry Schein</td><td>995-2949</td><td></td></tr><tr><td>Kwik Stop Styptic powder</td><td>Butler Schein</td><td>5867</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma</td><td>G8540</td><td></td></tr><tr><td>Methanol</td><td>Fisher</td><td>a452-4</td><td></td></tr><tr><td>Microfuge 22R Centrifuge</td><td>Bekman Colter</td><td>368831</td><td></td></tr><tr><td>Neosporin</td><td>Fisher</td><td>19-898-143</td><td></td></tr><tr><td>Neurobasal medium</td><td>Life Technologies</td><td>21103049</td><td></td></tr><tr><td>Nikon Stereoscope</td><td>Nikon</td><td>SMZ745T</td><td></td></tr><tr><td>Nucleospin Gel and PCR Cleanup</td><td>Machery-Nagel</td><td>740609</td><td></td></tr><tr><td>P200 pipet</td><td>Rainin</td><td>L-200XLS+</td><td></td></tr><tr><td>p24 Lenti-X rapid titer kit</td><td>Clontech</td><td>632200</td><td></td></tr><tr><td>PCR film seal</td><td>Fisher</td><td>AB0558</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma</td><td>P8340</td><td></td></tr><tr><td>ReFresh Charcoal Filter canister</td><td>E-Z Anesthesia</td><td>EZ-258</td><td></td></tr><tr><td>Scalpel blades, #10</td><td>Fine Science tools Inc</td><td>10010-00</td><td></td></tr><tr><td>SD rats 150-200 g</td><td>Charles River</td><td>Rats</td><td>rats ordered at 150-200 g.&amp;nbsp; Surgery 5 days after arrival</td></tr><tr><td>Small animal ear tags</td><td>National Band and Tag co</td><td>1005-1</td><td></td></tr><tr><td>Sterile surgical drapes</td><td>Braintree Scientific</td><td>SP-MPS</td><td></td></tr><tr><td>Synergy 2 plate reader</td><td>BioTek</td><td>n/a</td><td></td></tr><tr><td>TaqMan Universal PCR Master Mix</td><td>Applied Biosystems</td><td>4304437</td><td></td></tr><tr><td>Thapsigargin</td><td>Sigma</td><td>T9033</td><td>harmful to human health</td></tr><tr><td>Virapower lentiviral packaging mix</td><td>Life Technologies</td><td>K4975-00</td><td></td></tr><tr><td>Xfect Transfection reagent</td><td>Clontech</td><td>631318</td><td></td></tr><tr><td>Xylazine</td><td>Valley Vet</td><td>468RX</td><td></td></tr></tbody></table>

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.

The GLuc-SERCaMP method allows for assessment of ER calcium homeostasis by sampling extracellular fluids. Several controls can be included in the experimental design to enhance interpretation of results. First, use of a constitutively secreted reporter (e.g. GLuc without the C-terminal ASARTDL or &#8220;GLuc-No Tag&#8221;) can be employed to assess the effects of experimental treatments on the secretory pathway (global cellular secretion) and transgene expression. For instance, an increase in the extracellular l...

Watch this Scientific Journal Video about Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP at JoVE.com

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.

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 ...

monitoring-endoplasmic-reticulum-calcium-homeostasis-using-gaussia

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.1K Views.  National Institutes of Health. 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.

Video: Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Biochemistry

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, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

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

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 ...

A Simple Assay to Measure ER-Mitochondria Contacts for High-Throughput Screens

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describe&#160;a split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.

Author Spotlight: Regulation and Dysregulation of ER-Mitochondria Contacts &#8212; Implications for Neurodegenerative Disease Pathogenesis

We have established a split-luciferase reassembly assay to monitor the endoplasmic reticulum-mitochondria contacts in live cells. Using this assay, we describe a protocol to quantitatively measure the level of these inter-organelle couplings in HEK293T cells, under the condition of chemical treatment.

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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 ...

Neuroscience

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 ...

Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.

Author Spotlight: Investigating Viral Disruption of Intestinal Epithelial Signaling &#8211; Research Insights and Future Directions 

This protocol describes an approach for performing calcium imaging in virus-infected human intestinal organoids and offers an approach to analysis.

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory ...

Immunology and Infection

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+

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.

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.

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

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

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

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