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>

<ol>
	<li>Sitia, R., Braakman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+control+in+the+endoplasmic+reticulum+protein+factory.">Quality control in the endoplasmic reticulum protein factory.</a> Nature. 426 (6968), 891-894 (2003).</li><li>Burdakov, D., Petersen, O. H., Verkhratsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraluminal+calcium+as+a+primary+regulator+of+endoplasmic+reticulum+function.">Intraluminal calcium as a primary regulator of endoplasmic reticulum function.</a> Cell Calcium. 38 (3-4), 303-310 (2005).</li><li>Fu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+lipid+metabolism+disrupts+calcium+homeostasis+causing+liver+endoplasmic+reticulum+stress+in+obesity.">Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity.</a> Nature. 473 (7348), 528-531 (2011).</li><li>Micaroni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+calcium+in+intracellular+trafficking.">The role of calcium in intracellular trafficking.</a> Curr Mol Med. 10 (8), 763-773 (2010).</li><li>Mekahli, D., Bultynck, G., Parys, J. B., De Smedt, H., Missiaen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic-reticulum+calcium+depletion+and+disease.">Endoplasmic-reticulum calcium depletion and disease.</a> Cold Spring Harb Perspect Biol. 3 (6), (2011).</li><li>Paredes, R. M., Etzler, J. C., Watts, L. T., Zheng, W., Lechleiter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+calcium+indicators.">Chemical calcium indicators.</a> Methods. 46 (3), 143-151 (2008).</li><li>Whitaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+probes+for+measurement+of+intracellular+calcium.">Genetically encoded probes for measurement of intracellular calcium.</a> Methods Cell Biol. 99, 153-182 (2010).</li><li>Tang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+application+of+a+class+of+sensors+to+monitor+Ca2++dynamics+in+high+Ca2++concentration+cellular+compartments.">Design and application of a class of sensors to monitor Ca2+ dynamics in high Ca2+ concentration cellular compartments.</a> Proc Natl Acad Sci U S A. 108 (39), 16265-16270 (2011).</li><li>Palmer, A. E., Jin, C., Reed, J. C., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-2-mediated+alterations+in+endoplasmic+reticulum+Ca2++analyzed+with+an+improved+genetically+encoded+fluorescent+sensor.">Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor.</a> Proc Natl Acad Sci U S A. 101 (50), 17404-17409 (2004).</li><li>Suzuki, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intraorganellar+Ca2++at+subcellular+resolution+using+CEPIA.">Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA.</a> Nat Commun. 5, 4153 (2014).</li><li>Rehberg, M., Lepier, A., Solchenberger, B., Osten, P., Blum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+non-disruptive+strategy+to+target+calcium+indicator+dyes+to+the+endoplasmic+reticulum.">A new non-disruptive strategy to target calcium indicator dyes to the endoplasmic reticulum.</a> Cell Calcium. 44 (4), 386-399 (2008).</li><li>Henderson, M. J., Wires, E. S., Trychta, K. A., Richie, C. T., Harvey, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SERCaMP:+a+carboxy-terminal+protein+modification+that+enables+monitoring+of+ER+calcium+homeostasis.">SERCaMP: a carboxy-terminal protein modification that enables monitoring of ER calcium homeostasis.</a> Mol Biol Cell. 25 (18), 2828-2839 (2014).</li><li>Henderson, M. J., Richie, C. T., Airavaara, M., Wang, Y., Harvey, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesencephalic+astrocyte-derived+neurotrophic+factor+(MANF)+secretion+and+cell+surface+binding+are+modulated+by+KDEL+receptors.">Mesencephalic astrocyte-derived neurotrophic factor (MANF) secretion and cell surface binding are modulated by KDEL receptors.</a> J Biol Chem. 288 (6), 4209-4225 (2013).</li><li>Shipman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+4-(2-hydroxyethyl)-1-piperazine&#235;thanesulfonic+acid+(HEPES)+as+a+tissue+culture+buffer.">Evaluation of 4-(2-hydroxyethyl)-1-piperazine&#235;thanesulfonic acid (HEPES) as a tissue culture buffer.</a> Proc Soc Exp Biol Med. 130 (1), 305-310 (1969).</li><li>Zigler, J. S., Lepe-Zuniga, J. L., Vistica, B., Gery, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+cytotoxic+effects+of+light-exposed+HEPES-containing+culture+medium.">Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium.</a> In Vitro Cell Dev Biol. 21 (5), 282-287 (1985).</li><li>Howard, D. B., Powers, K., Wang, Y., Harvey, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tropism+and+toxicity+of+adeno-associated+viral+vector+serotypes+1,+2,+5,+6,+7,+8,+and+9+in+rat+neurons+and+glia+in+vitro.">Tropism and toxicity of adeno-associated viral vector serotypes 1, 2, 5, 6, 7, 8, and 9 in rat neurons and glia in vitro.</a> Virology. 372 (1), 24-34 (2008).</li><li>Smeets, E. F., Heemskerk, J. W., Comfurius, P., Bevers, E. M., Zwaal, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thapsigargin+amplifies+the+platelet+procoagulant+response+caused+by+thrombin.">Thapsigargin amplifies the platelet procoagulant response caused by thrombin.</a> Thromb Haemost. 70 (6), 1024-1029 (1993).</li><li>Tannous, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaussia+luciferase+reporter+assay+for+monitoring+biological+processes+in+culture+and+in+vivo.">Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo.</a> Nat Protoc. 4 (4), 582-591 (2009).</li><li>Sobrevals, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+vectors+transduce+hepatocytes+in+vivo+as+efficiently+in+cirrhotic+as+in+healthy+rat+livers.">AAV vectors transduce hepatocytes in vivo as efficiently in cirrhotic as in healthy rat livers.</a> Gene Ther. 19 (4), 411-417 (2012).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+highly+efficient+transduction+by+double-stranded+adeno-associated+virus+vectors+in+vitro+and+in+vivo.">Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo.</a> Gene Ther. 10 (26), 2105-2111 (2003).</li><li>Seppen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+vector+serotypes+mediate+sustained+correction+of+bilirubin+UDP+glucuronosyltransferase+deficiency+in+rats.">Adeno-associated virus vector serotypes mediate sustained correction of bilirubin UDP glucuronosyltransferase deficiency in rats.</a> Mol Ther. 13 (6), 1085-1092 (2006).</li><li>Hareendran, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+(AAV)+vectors+in+gene+therapy:+immune+challenges+and+strategies+to+circumvent+them.">Adeno-associated virus (AAV) vectors in gene therapy: immune challenges and strategies to circumvent them.</a> Rev Med Virol. 23 (6), 399-413 (2013).</li></ol>

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1.5mL tubes</td>
			<td>Fisher&#160;</td>
			<td>02-682-550</td>
			<td></td>
		</tr>
		<tr>
			<td>10% NP-40 solution&#160;</td>
			<td>Pierce</td>
			<td>28324</td>
			<td>for intracellular GLuc assays</td>
		</tr>
		<tr>
			<td>1mL luer-lok syringes</td>
			<td>Fisher</td>
			<td>14-823-30</td>
			<td></td>
		</tr>
		<tr>
			<td>200uL 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>30g needles</td>
			<td>Fisher Scientific</td>
			<td>14-821-13A&#160;</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>1:2000 (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&#39;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 150-200g</td>
			<td>Charles River</td>
			<td>Rats</td>
			<td>rats ordered at 150-200g.&#160; 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

Research

JoVE Journal

Biology

13.0K 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

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

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, ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

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 ...