Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.
The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic 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 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’s, and Parkinson’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.
1. In Vitro Assay: Detecting SERCaMP Release from a Stable SH-SY5Y Cell Line
2. In Vitro 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.
3. In Vitro Assay: Viral Vector-mediated Expression of GLuc-SERCaMP
Note: Adeno-associated viral (AAV) vector packaging16 and purification12 and lentivirus production13 have been previously reported.
4. In Vivo SERCaMP Assay
Note: Before conducting 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.
5. Luminescence Assay
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 “GLuc-No Tag”) 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 levels of both GLuc-SERCaMP and GLuc-No Tag would be considered an ambiguous result. Alternatively, an increase in GLuc-SERCaMP secretion with a corresponding lack of GLuc-No Tag response supports an ER calcium dependent event (Figure 8). The secretion of GLuc-SERCaMP in response to ER calcium depletion can be further assessed by measuring intracellular GLuc, although this is limited to a single timepoint as lysis is required. Intracellular GLuc-SERCaMP will decrease in response to ER calcium depletion, as it is progressively secreted, and the extracellular:intracellular ratio (calculated for each individual well) will increase (Figure 2B). The extracellular:intracellular ratio is useful to control for changes in transgene expression.
Pharmacologic modulators of the ER calcium store can be utilized to confirm the contribution of ER calcium to SERCaMP release in novel paradigms. Dantrolene, a RyR antagonist, is known to stabilize ER calcium, which should reduce or inhibit the release of SERCaMP in response to Tg (Figure 8B). It is recommended, when possible, to test additional compounds that affect ER calcium flux (e.g. Xestospongin C, 4-chloro-m-cresol) in novel experimental paradigms employing SERCaMP. These studies are often challenging in vivo, but may be feasible in corresponding tissue culture models.
For in vitro studies using GLuc-based SERCaMP, is recommended to calculate responses on individual plates relative to the control(s). The ‘treatment-induced’ response can be assessed over the timecourse by tracking changes in relative response versus control (for between-plate comparisons). It is not advisable to compare raw numbers across multiple plates, owing to decay of substrate, which can lead to changes in the raw values between timepoints (Figure 6C). Making relative comparisons within a plate minimizes this effect (Figure 6D). An example of data analysis for an in vitro Tg-induced SERCaMP release experiment is presented in Figure 9.
For in vivo studies using GLuc-SERCaMP, data should be assessed independently for each animal, with every animal serving as its own ‘pre-treatment’ control. This is necessary to account for variability in transgene delivery and expression between animals (Figure 4A).
The magnitude of SERCaMP responses will be affected by a variety of factors, many of which are related to baseline health of the cells and can be directly controlled by the investigator. For maximal SERCaMP responsiveness, it is imperative to optimize conditions that favor basal ER calcium homeostasis. This includes seeding cells at optimal density and using low viral titers. Different cell and tissue types may have additional requirements, which should be determined empirically.
Figure 1: Stable SH-SY5Y cell line seeding density and passage number considerations. (A) SH-SY5Y-GLuc-SERCaMP cells were seeded at various densities and incubated for 40 hr. Secreted GLuc-SERCaMP was measured 4 hr after treatment with 300 nM Tg or vehicle control (mean ± SD, n = 6). Thapsigargin-induced increase in GLuc-SERCaMP secretion is indicated for each cell density. (B) SH-SY5Y-GLuc-SERCaMP cells at passage number 2 and 15 were examined for thapsigargin-induced secretion. Cells were treated with 100 nM Tg for 2 hr or 4 hr, and secreted GLuc activity was normalized to vehicle treated controls (mean ± SD, n = 4, no significant effect of passage-factor, 2-way ANOVA). Dashed line indicates y = 1. Please click here to view a larger version of this figure.
Figure 2: Enzymatic assay for measurement of intracellular GLuc-SERCaMP. (A) Intracellular and (B) extracellular GLuc enzymatic activity was assessed in rat primary cortical neurons (transduced with AAV-GLuc-SERCaMP). Six days after transduction, cells were treated with 60 nM thapsigargin for 4 hr. As GLuc-ASARTDL accumulates in the medium, corresponding decreases in intracellular levels are observed. (C) A ratio of extracellular: intracellular GLuc activity can be calculated for each individual well.
Figure 3: AAV-GLuc-ASARTDL titer versus thapsigargin-induced response for SH-SY5Y cells and rat primary cortical neurons. (A) SH-SY5Y cells were seeded in 96 well plates at 5 x 104 cells per well and allowed to adhere overnight. Cells were transduced at the indicated multiplicity of infection (MOI), incubated for 48 hr and then treated with 300 nM Tg or vehicle. Luminescence was measured in the medium after 8 hr (mean ± SEM, n = 3). * p <0.05, multiple t-tests (Holm-Sidak correction). (B) Rat primary cortical neurons were transduced on DIV8 with AAV-GLuc-ASARTDL at the indicated MOI (calculated using 60,000 cells per well approximation at time of transduction). Five days after transduction, cells were treated with 100 nM Tg or vehicle control and luminescence in the medium was measured after 8 hr (mean ± SEM, n = 6). * p <0.05, multiple t-tests (Holm-Sidak correction).
Figure 4: Thapsigargin-induced GLuc-SERCaMP release into blood following intrahepatic injections over range of viral titers. (A) Raw luminescence values from rats (n = 8) intrahepatically injected with different AAV-GLuc-ASARTDL titers. Thapsigargin (1 mg/kg) injection (i.p.) was administered on day 12. Blood was collected at indicated time points and stored at -80 °C until time of assay. (B) Normalized luminescence values from panel A, demonstrating thapsigargin-induced SERCaMP response in rats (n = 8) expressing various amounts of AAV-GLuc-ASARTDL.
Figure 5: Handling and storage of GLuc-SERCaMP plasma samples (in vivo). (A) Plasma was collected from rats intrahepatically expressing GLuc-SERCaMP (n = 10), aliquoted and stored at -80 °C until time of assays. Tubes were removed from -80 °C and stored at 4 °C for indicated times prior to luminescence measurement (mean ± SD). (B) Effect of multiple freeze/thaws of plasma samples on GLuc enzymatic activity. Plasma samples collected from rats intrahepatically expressing AAV-GLuc-SERCaMP (n = 10) were subjected to the indicated number of freeze-thaw cycles and assayed for luminescence (mean ± SD).
Figure 6: Preparing coelenterazine substrate for GLuc-SERCaMP assays. (A) Culture medium was collected from SH-SY5Y-GLuc-SERCaMP cells lines treated with 300 nM Tg (or vehicle control) for 5 hr. GLuc activity in the medium was assessed by injecting PBS + various concentrations of coelenterazine (mean ± SD, n = 6). (B) Luminescence values from panel A were normalized to vehicle treated control at each substrate concentration to assess the thapsigargin-induced effect. (C) Substrate decay over several hours. Luminescence from a known amount of recombinant GLuc (0.1 ng or 0.02 ng) was measured over several hours after preparing substrate (mean ± SD, n = 4). (D) While the raw luminescence for the recombinant GLuc decreased over time due to substrate decay, the fold difference in the samples (as determined by luminescence) was maintained.
Figure 7: Considerations for GLuc-SERCaMP assays related to kinetics of GLuc/CTZ reaction. (A) Culture medium alters the decay of GLuc/CTZ signal. 5 µl of supernatant containing GLuc-SERCaMP was mixed with varying ratios of PBS and culture medium. 100 µl of substrate (15 µM CTZ in PBS + 500 mM ascorbic acid) was added to 50 µl of sample that was diluted as outlined in the table. Light emission was monitored over 15 min. (B) 5 µl of medium was collected from SH-SY5Y-GLuc-ASARTDL cells and 100 µl substrate (8 µM CTZ in PBS + 5 mM NaCl) was added. Luminescence was measured immediately after adding substrate (T = 0) and every 30 sec over the course of 15 min (± SD, n = 12). Data is plotted as the percent signal relative to the previous 30 sec interval to assess the rate of decay. The inset shows the raw luminescence data.
Figure 8: Pharmacologic corroboration of ER calcium depletion. (A) Effect of 100 nM thapsigargin on GLuc secretion was examined for GLuc-ASARTDL and GLuc-No Tag control (mean ± SD, n = 6). (B) Stabilizing ER calcium with dantrolene (RyR antagonist) inhibits Tg-induced SERCaMP release. Cells were pre-treated with the indicated concentrations of dantrolene for 30 min or 16 hr before addition of 100 nM thapsigargin. GLuc activity in the medium was measured after 4 hr (mean ± SD, n = 6).
Figure 9: Mock representative results for the GLuc-SERCaMP assay. Note the vehicle treated values may decrease between plates, due to substrate breakdown (dependent on time interval between reading individual plates). To account for this effect, the treatment specific effect is calculated independently for each plate and this ratio is compared across plates.
Date | Rat | Tube mass (mg) | Heparin volume (ml) | Heparin density (g/ml) | heparin mass (mg) | Mass of tube + heparin (mg) | Total mass after blood collection (mg) | Mass of collected blood (mg) | Density blood (g/ml) | Calculated volume blood (ul) | Volume of heparin needed for 2:1 ratio | ml additional heparin to add before spin |
01.01.15 | SD Male | 1135.9 | 50 | 1.117 | 55.83 | 1191.73 | 1317.70 | 125.97 | 1.05 | 119.97 | 59.98 | 9.98 |
Table 1: Blood collection table. Example of blood collection log for SERCaMP studies.
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 cortical neurons, SH-SY5Y cells, and rat liver (Figures 3 and 4). We previously described the generation of a stably transfected cell line expressing GLuc-ASARTDL and demonstrated that as few as 20 cells in a population of 5 x 104 unlabeled cells can report a thapsigargin induced response12. Here we show that the stable cell line can consistently report Tg-induced response out to 15 passages, the most analyzed, indicating that continuous expression of the reporter over time does not impact its ability to be released in response to ER calcium depletion (Figure 1). Our data indicate that GLuc-SERCaMP is primarily held within the ER of a cell until a time of ER calcium depletion when it is secreted. Under “normal” conditions, there is a low basal secretion that allows the establishment of baseline ER calcium homeostasis12. Following ER calcium depletion by thapsigargin, levels of intracellular and extracellular luminescence change in opposite directions. This can be expressed as a ratio to indicate a shift in steady state distribution of the sensor (Figure 2). Importantly, as SERCaMP release is modulated by KDEL receptor expression12, the magnitude of release may vary depending on cell type. We envision that substituting ‘ASARTDL’ with alternate KDEL-like carboxy-terminal sequences may be required to achieve maximal SERCaMP response in a specific cell or tissue type.
For in vitro assays, levels of extracellular GLuc-SERCaMP will accumulate over time due to the stability of the secreted reporter; we reported an approximate 5-10% decrease in activity after 72 hr12. As such, exchanging medium prior to initiating a pharmacologic or genetic challenge to ER calcium homeostasis may be necessary to reduce background signal due to sensor accumulation. In contrast, the half-life of GLuc-SERCAMP in vivo is 3.5-4.7 min12 indicating signal in plasma represents a recent release of the reporter into circulating blood. Once blood is ex vivo and processed to plasma, however, GLuc-SERCaMP is very stable (Figure 5).
For the methodologies described, medium or plasma is transferred to opaque 96-well plates before enzymatic assays. Two important factors to consider when using GLuc-based reporters are the enzyme’s flash kinetics and breakdown of the coelenterazine substrate. Plate readers equipped with injectors are best suited for running GLuc enzymatic assays to normalize the time between substrate addition and luminescence measurements. The chemical properties of coelenterazine make it prone to decay, which precludes comparing raw luminescence values measured at different times after substrate preparation. Fold differences among samples, however, are maintained (Figure 6), allowing the relative difference between control and experimental samples to be tracked over the duration of experiments (Figure 9).
The ability to monitor ER calcium fluctuations in vivo is advantageous when investigating progressive diseases. While other methods, such as fluorescent cytoplasmic dyes, are suitable for acute in vitro studies, a SERCaMP reporter is the first to allow longitudinal ER calcium monitoring. Our protocol outlines direct hepatic injections of AAV-GLuc-SERCaMP. We have detected steady levels of GLuc-SERCaMP in circulation of unchallenged animals for 56 days post-injection (latest timepoint tested thus far) and predict longer experiments are possible12. AAV vectors are small in size, measuring approximately 20 nm in diameter, and pose minimal biosafety requirements19. To circumvent the conversion of AAV single-stranded genome to double-stranded DNA, AAV-SERCaMP was packaged as an AAV serotype-1 double-stranded vector20. AAV serotype-1 has been shown to effectively transduce rat livers 19,21; however, the caveat of our injection technique is the potential for virus to travel to other tissues throughout the body. Although the vector was directly injected into the liver, our methodology as presented cannot discern the source of SERCaMP release, and therefore it is possible that tissues other than the liver may contribute to the GLuc-SERCaMP signal. Future genetic manipulations will restrict expression to target tissue types. For example, tissue-specific Cre driver lines crossed with a Cre-dependent GLuc-SERCaMP will allow for tissue-specific monitoring of ER calcium via plasma sampling.
The described in vivo technique uses low viral titers due to the robust nature of the reporter. GLuc-SERCaMP release can be detected from a viral range of 7.6 x 107 vg to 7.6 x 109 vg (Figure 4). This range is 4-400 times lower than reported in previous work utilizing firefly luciferase-based viral injections of the liver19. At higher concentrations, we observed a loss of detectable expression over time, which is possibly due to an immune response of the animal to the transgene22. Higher titers of virus should be avoided when possible due to increased likelihood of signal loss. Lower titers than those presented have not been tested. This protocol outlines injections of AAV-GLuc-SERCaMP into liver; similar approaches are in theory feasible for other tissue types. Parameters such as viral concentration and serotype must be optimized for sufficient expression in other tissues.
Due to the robust nature of the reporter, small volumes of blood (100-150 µl) can be collected from tail clippings, allowing for repeated collections throughout experiments. This was useful for longitudinal characterization of GLuc-SERCaMP release in response to thapsigargin, where we observed elevated levels after thapsigargin administration followed by a return to baseline levels (Figure 4). Proper handling of the plasma samples following blood collection is also important. As such, we have demonstrated that SERCaMP is stable in plasma stored at 4 °C up to 72 hr prior to enzymatic assay (Figure 5). Furthermore, plasma samples can undergo at least three freeze/thaw cycles with minimal effect on luminescence (Figure 5). In practice, we store all samples at -80 °C, avoid unnecessary freeze-thaw cycles prior to assessing enzymatic assay, and analyze all samples for an experiment at the same time.
ER calcium depletion is implicated in the pathogenesis of a variety of diseases. It is often thought to be an upstream event that hinders cellular functions and leads to the activation of the unfolded protein response (UPR). The UPR is an adaptive response employed by the ER to reestablish homeostatic conditions5. Chronic states of ER stress exceed the capacity of the UPR to restore homeostasis, ultimately leading to cell death. ER stress and ER calcium dysregulation are observed in diseases such as type 1 diabetes, diabetic nephropathy, neurodegenerative diseases and cardiovascular dieases5. The relationship between calcium dysregulation and disease pathogenesis, however, is difficult to delineate. The SERCaMP technology has the potential to track this process over the lifespan of an animal, thus providing insight into disease development and progression. Moreover, the evaluation of potential therapeutics designed to prevent or correct ER calcium dysregulation can be assessed through the use of SERCaMP. Lastly, identifying diseases where GLuc-SERCaMP release occurs offers the opportunity to engineer and employ secreted therapeutic proteins or peptides as SERCaMPs as a means of disease regulated gene therapy.
The authors have nothing to disclose.
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.
1.5mL tubes | Fisher | 02-682-550 | |
10% NP-40 solution | Pierce | 28324 | for intracellular GLuc assays |
1mL luer-lok syringes | Fisher | 14-823-30 | |
200uL filter tips | Rainin | RT-L200F | |
3-0 surgical sutures | Fisher | NC9598192 | |
30g needles | Fisher Scientific | 14-821-13A | |
Adhesive microplate sealing sheets | Thermo | AB-0558 | |
Alcohol prep pads | Fisher | 22-246-073 | |
Anesthesia Auto Flow System | E-Z Anesthesia | EZ-AF9000 | |
Animal recovery chamber | Lyon Vet | ICU-912-004 | |
B27 supplement | Life Technologies | 17504-044 | |
Betadine solution | Fisher | NC9386574 | |
Bleach | Clorox | n/a | |
Bovine growth serum | Thermo | SH30541.03 | |
Coelenterazine, Native | Regis Technologies | 1-361204-200 | |
Cotton tipped applicators | Puritan | 806-WC | |
Cutting needles 3/8 circle sutures | WPI | 501803 | |
Digital ultrasconic cleaner | Fisher Scientific | FS60D | |
DMEM high glucose, GlutaMAX, pyruvate | Life Technologies | 10569-010 | |
DNA mass ladder | Life Technologies | 10496-016 | |
Gaussia luciferase (recombinant protein) | Nanolight | 321-100 | |
Gaussia luciferase antibody (for WB, ICC, or IHC) | New England Biolabs | E8023S | 1:2000 (WB) |
Germinator 500 | CellPoint Scientific | DS-401 | |
Gluc assay plates (96 well, opaque) | Fisher | 07-200-589 | |
Hank's balanced salt solution | Life Technologies | 14175-095 | |
Heparin | Allmedtech | 63323-276-02 | |
Isoflurane | Butler Schein | 29404 | |
Ketamine | Henry Schein | 995-2949 | |
Kwik Stop Styptic powder | Butler Schein | 5867 | |
L-glutamine | Sigma | G8540 | |
Methanol | Fisher | a452-4 | |
Microfuge 22R Centrifuge | Bekman Colter | 368831 | |
Neosporin | Fisher | 19-898-143 | |
Neurobasal medium | Life Technologies | 21103049 | |
Nikon Stereoscope | Nikon | SMZ745T | |
Nucleospin Gel and PCR Cleanup | Machery-Nagel | 740609 | |
P200 pipet | Rainin | L-200XLS+ | |
p24 Lenti-X rapid titer kit | Clontech | 632200 | |
PCR film seal | Fisher | AB0558 | |
Penicillin/streptomycin | Life Technologies | 15140-122 | |
Protease inhibitor cocktail | Sigma | P8340 | |
ReFresh Charcoal Filter canister | E-Z Anesthesia | EZ-258 | |
Scalpel blades, #10 | Fine Science tools Inc | 10010-00 | |
SD rats 150-200g | Charles River | Rats | rats ordered at 150-200g. Surgery 5 days after arrival |
Small animal ear tags | National Band and Tag co | 1005-1 | |
Sterile surgical drapes | Braintree Scientific | SP-MPS | |
Synergy 2 plate reader | BioTek | n/a | |
TaqMan Universal PCR Master Mix | Applied Biosystems | 4304437 | |
Thapsigargin | Sigma | T9033 | harmful to human health |
Virapower lentiviral packaging mix | Life Technologies | K4975-00 | |
Xfect Transfection reagent | Clontech | 631318 | |
Xylazine | Valley Vet | 468RX |