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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

An improved protocol for a calcium mobilization assay with endothelial cells, used to identify ligands of protease-activated receptors (PARs), has been developed. The new protocol reduces total assay time by 90-120 min and yields reproducible concentration-response curves.

Abstract

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of Gαq signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.

Introduction

Protease-activated receptors (PARs)1,2,3 are a subfamily of class A G protein-coupled receptors (GPCRs) that are expressed in a variety of cell types, including platelets and endothelial cells4,5,6,7. Unlike the majority of GPCRs, PARs have a unique intramolecular mode of activation. Most GPCRs are activated by soluble ligands interacting with a distinct binding pocket, but PARs are activated by the proteolytic cleavage of the N-terminus, which results in a new tethered ligand that can interact with the extracellular loop 2 domain on the surface of a cell6,8,9. This interaction activates the receptor and can initiate several signaling pathways, promoting effects such as inflammation and platelet activation4,10,11,12. Different proteases can activate PARs through cleavage at unique sites on the N-terminus, revealing different tethered ligands (TL) that stabilize receptor conformations, which initiate different signaling pathways9,13,14,15. For example, in the most well-studied member of the subfamily, PAR1, cleavage by thrombin is used to support numerous biological processes, including platelet activation and leukocyte recruitment to the endothelium, but can lead to deleterious effects when the receptor is overexpressed or overactivated4,16,17,18,19,20,21. Conversely, cleavage by activated protein C (aPC) can promote anti-inflammatory effects and maintenance of endothelial barriers15,22,23,24,25,26,27,28,29. PARs can also be activated by peptide analogs of the TLs in an intermolecular fashion13,30,31. These peptides are routinely used to measure PAR inhibition (modulation) in place of PAR-targeting proteases, and they are used in this protocol.

Numerous disorders are associated with pathological PAR1 signaling, including sepsis22,32, cardiovascular disease33,34,35,36,37,38, kidney disease39,40,41,42, sickle cell disease43, fibrosis44, osteoporosis and osteoarthritis45,46, neurodegeneration47,48,49,50,51, and cancer52,53,54,55,56,57,58,59. Antagonists of PAR1 have been studied since the 1990s as antiplatelet agents for cardiovascular disease, and the growing list of diseases associated with the receptor necessitates the identification of novel ligands for use as biological probes (tool compounds) or as potential therapeutics. Currently, there is only one FDA-approved PAR1 antagonist, vorapaxar, which is used to treat coronary artery disease in high-risk patients34,36,37,60. An alternative PAR1 antagonist, the pepducin PZ-128, completed a successful phase II study to prevent thrombosis in cardiac catheterization patients38. The Dockendorff group has focused on the medicinal chemistry and pharmacology of a separate class of small molecules, PAR1 ligands known as parmodulins61,62. Unlike reported PAR1 antagonists such as vorapaxar, parmodulins are allosteric, biased modulators of PAR1 that selectively block the Gαq pathway while promoting cytoprotective effects similar to aPC. Unlike potent orthosteric PAR1 antagonists such as vorapaxar, published parmodulins are also reversible63,64,65.

Initially, parmodulins were identified by Flaumenhaft and coworkers for their ability to inhibit P-selectin expression or granule secretion in platelets61,66. However, an alternative method was required to study the effects of parmodulins on endothelial cells. One common method to monitor GPCR-related signaling is to measure intracellular Ca2+ mobilization, an important secondary messenger that can be measured using a suitable intracellular calcium-binding dye67,68. Substantial evidence has been provided showing that calcium mobilization induced by PAR1 is through the activation of Gαq69,70. Once activated by its tethered ligand (or a suitable exogenous ligand), PAR1 undergoes a conformational change which causes guanosine diphosphate (GDP) bound to the Gαq subunit to be replaced by guanosine triphosphate (GTP)68. The Gαq subunit then activates phospholipase Cβ (PLC-β), which catalyzes the hydrolysis of phosphatidylinositol 4,5 bisphosphate (PIP2), forming 1,4,5-inositol triphosphate (IP3) and diacylglycerol (DAG). Finally, IP3 binds to IP3-sensitive Ca2+ channels in the membrane of the endoplasmic reticulum, allowing Ca2+ to be released into the cytoplasm, where it can bind to Ca2+-dependent fluorescent dyes, such as Fluo-4, that are added to the cells71. This process occurs within seconds and can increase the concentration of Ca2+ 100-fold, leading to a drastic change in the amount of calcium-bound dye and a robust fluorescence signal.

In 2018, the Dockendorff group disclosed a medium-throughput Ca2+ mobilization assay that could be used to identify antagonists of the Gαq pathway of PAR172. The assay used EA.hy92673, a hybrid human endothelial cell line, which can be used for multiple passages without a noticeable change in PAR1 expression, and is established for in vitro measurements of cytoprotective effects. 

The original protocol used EA.hy926 cells in 96-well plates and loaded with Fluo-4/AM dye, which was chosen due to its intense fluorescence at 488 nm and high cell permeability. Once the dye was loaded into the cells, lengthy washing steps were performed with an automated 8-channel liquid handler (faster methods of liquid handling, such as a 96-channel washer, were inaccessible). The reproducibility of this assay was superior to that without the careful, automated robotic media changes. Antagonists were then incubated with the cells, PAR1 was activated through the sequential addition of a selective agonist (16 wells at a time), and changes in fluorescence resulting from calcium mobilization and dye binding were measured to determine activity.

While this protocol allows for the measurement of PAR1-mediated calcium mobilization, it is limited by the time required to assay each 96-well plate. Long experiment times are problematic not only because the number of compounds that can be screened each day is limited, but also because dye efflux occurs over time, narrowing the assay window by increasing the basal fluorescence. One contributor to the long experiment time is the use of an 8-channel liquid handler for plate washing, which adds over 30 min to each experiment. The required tips also became difficult to obtain due to supply chain problems. Here an updated protocol for the PAR-mediated calcium mobilization assay that does not require a liquid handler, and therefore can be run in higher throughput, is reported. This protocol should also be suitable for measuring signaling with other GPCRs that lead to intracellular calcium mobilization. This updated plate reader protocol is ideal for academic and small industrial labs that do not have the resources for expensive cell imaging instruments but have a need to rapidly screen numerous compounds. For an example of a calcium mobilization assay using a plate imager, see Caers et al.74.

Protocol

All media exchanges/additions made in steps 1 and 2 of the following protocol are performed in a sterile hood. Unless otherwise noted, all plasticware used in the sterile hood must be purchased sterilized and sealed or sterilized appropriately via autoclave.

1. Initiation of EA.hy926 cell line

  1. Acquire EA.hy926 cells.
  2. Store vial(s) of cells in the vapor phase of a liquid nitrogen tank.
  3. Prepare DMEM complete medium by first warming DMEM, FBS, and pen/strep (see Table of Materials for specific details) in a 37 °C water bath for 15-30 min. Add 50 mL of FBS and 1 mL of pen/strep to 500 mL of DMEM. Gently mix the solution by inversion.
    NOTE: Mixing too vigorously causes excessive amounts of bubbles.
  4. Remove one vial of cells from liquid nitrogen and warm in a 37 °C water bath for 5 min.
  5. Sterilize the vial of cells and bottle of DMEM complete medium by spraying with alcohol, then move to the sterile hood.
  6. Use a 1,000 µL pipette to carefully transfer the cells to a 15 mL sterile centrifuge tube. Add 1 mL of DMEM to the vial to rinse and add to the centrifuge tube.
  7. Make a pellet of cells using a centrifuge (150 × g, 5 min, 22 °C). Remove the medium from the centrifuge tube by aspiration; be careful not to disturb the cell pellet.
  8. Add 3 mL of DMEM complete medium to the centrifuge tube and gently break apart the pellet by mixing the cell suspension using a 1,000 µL pipette.
    NOTE: Causing bubbles by mixing too vigorously should be avoided as this could activate or damage the cells.
  9. Mix 10 µL of the cell suspension with 10 µL of trypan blue in a microcentrifuge tube using a pipette with either 20 µL or 200 µL tips. Add 10 µL of the cell suspension/trypan blue mixture to a hemocytometer and count the cells.
    NOTE: Using 0.1-10 µL pipette tips can shear the cells. Tips of this size should not be used while handling the cell suspension.
  10. Add DMEM complete medium to the cell suspension to make a density of 66,000 cells/mL using either a 10 or 25 mL serological pipette. Pipette 15 mL of the cell suspension into T-75 culture flask(s). Ensure equal dispersion of the cell suspension by moving the flask north to south and east to west.
  11. Incubate the cells until they reach confluency (typically after 4-6 days), as estimated by examination under a microscope. At this point, use the cells in the experiment (beginning with section 2) or seed them into new flasks for expansion to prepare frozen stocks (at a recommended seeding density of 1,000,000 cells in 15 mL of complete medium per T-75 flask).
    NOTE: Cells can be grown in the culture flasks for as long as 3 weeks without a noticeable difference in shape, health, or performance in the assay. Complete medium should be exchanged every 3-4 days.

2. Addition of EA.hy926 cells to 96-well plates

NOTE: Cells from one confluent T-75 culture flask can be used to prepare two or three assay plates or optionally expanded into as many as 15 fresh T-75 flasks. At least five assay plates can be screened using the protocol in sections 4 and 5 in a normal workday. The following instructions describe preparing and testing one assay plate, but additional plates can be prepared by repeating steps 2.2, 2.7, 2.12, and 3.2 to prepare the desired number of assay plates. Most commonly, four assay plates are prepared per day to measure concentration-responses with up to 16 compounds, which requires two T-75 flasks with confluent cells.

  1. Warm DMEM complete medium in a 37 °C water bath for 15-30 min. Sterilize the bottle of DMEM complete medium by spraying it with alcohol, then move it to the sterile hood.
  2. Add 100 µL of a sterilized 0.4% gelatin solution to all wells of a black-walled, 96-well, clear-bottom plate with a lid. Incubate the plate with the gelatin solution in an incubator (37 °C, 5% CO2) for 30 min.
  3. Confirm that cells in a T-75 culture flask are 80-100% confluent using an inverted microscope. Remove DMEM complete medium from the T-75 flask via aspiration.
  4. Wash the cells in the flask with 10 mL PBS and gently move the flask North to South and East to West. Remove the PBS from the flask via aspiration.
  5. Add 5 mL of cell dissociation solution to the flask and incubate the flask in an incubator (37 °C, 5% CO2) for ~12 min. Tap the flask after 6 min to help facilitate dissociation.
    NOTE: Incubating cells for longer than 15 min with the referenced cell dissociation agent causes cells to begin to clump together, which makes obtaining an accurate cell count more difficult and may interfere with the assay.
  6. Add 5 mL of the prewarmed DMEM complete medium to the flask and gently mix into the cell dissociation solution/cell suspension. Using a 10 mL serological pipette, add the cell suspension into a sterile 50 mL centrifuge tube. Make a pellet of cells using a centrifuge (150 × g, 5 min, 22 °C).
  7. While the pellet is being formed, remove the gelatin solution from the 96-well plate via aspiration using a Pasteur pipette connected to a vacuum. Remove the plate lid from the time of gelatin removal until the solution is ready to be plated.
    NOTE: A multichannel pipette or sterile manifold connected to a vacuum may optionally be used.
  8. Remove the medium from the centrifuge tube by aspiration using a Pasteur pipette connected to a vacuum or a serological pipette; be careful not to disturb the cell pellet.
  9. Add fresh DMEM complete medium to the centrifuge tube using a 5 or 10 mL serological pipette. Gently break up the cell pellet by mixing the DMEM complete medium with a 1,000 µL pipette or a 5 mL serological pipette.
    NOTE: Eight milliliters of DMEM are typically used, but higher volumes can be added to make cell counting easier.
  10. Mix 10 µL of the cell suspension with 10 µL of trypan blue in a microcentrifuge tube using a pipette with either 20 µL or 200 µL tips. Add 10 µL of the cell suspension/trypan blue mixture to a hemocytometer and count the cells.
  11. Add DMEM complete media to the cell suspension to make a density of 600,000 cells/mL using either a 10 or 25 mL serological pipette. This will give a final density of 60,000 cells/well in a 96-well plate (determined to be the optimal density by experimentation).
    NOTE: A normal successful cell culture will yield between 12,000,000 and 15,000,000 cells, which can seed two to three 96-well plates.
  12. Mix the cell suspension by gently inverting the centrifuge tube and add it to a 50 mL multi-channel pipette reservoir. Mixing the suspension in the reservoir every 30 s by gently rocking the reservoir side to side, add 100 µL of the cell suspension to each well using a multi-channel pipette. Replace the transparent plate cover and gently shake the plate by sliding it on the surface of the sterile hood from North to South and East to West to ensure even cell distribution. Incubate the plate in the incubator (37 °C, 5% CO2) for 16-24 h.
    NOTE: Additional cell suspension can be used to prepare additional plates, or steps 1.10-1.11 can be repeated to continue the cell line.

3. Calcium mobilization assay preparation

  1. Prepare assay reagents.
    1. Prepare probenecid solution (250 mM, aqueous) by adding 36 mg of probenecid to a microcentrifuge tube and dissolving it in 0.6 M NaOH (500 µL). Vortex the solution.
      NOTE: Probenecid improves the retention of dye in the cell by inhibiting organic ion transporters75,76,77.
    2. To make HBSS-HEPES assay buffer, add HEPES (1.19 g) to a 500 mL bottle of Ca/Mg/phenol red-free HBSS (making a 10 mM solution of HEPES). Supplement the buffer with MgCl2 (1 M aqueous solution, 500 µL) and CaCl2 (1 M aqueous solution, 500 µL). Mix the solution and store in a refrigerator at 5 °C when not in use.
      1. For each 96-well plate, add 50 mL of the HBSS-HEPES assay buffer to a 50 mL centrifuge tube, supplement with 500 µL of the probenecid solution, and mix well. Warm the buffer to room temperature prior to use.
    3. Make a 10% Pluronic F-127 solution in DMSO by adding Pluronic F-127 (20 mg) to a microcentrifuge tube or HPLC vial and dissolving it with 200 µL of DMSO.
      NOTE: This solution can be stored at room temperature and is sufficient for about 30 plates. Pluronic F-127 dissolves very slowly at room temperature and should be gently heated to facilitate dissolution.
    4. Prepare the dye loading buffer by first adding 24 µL of DMSO to a vial of Fluo-4/AM (50 µg) to dissolve the dye. In a foil-wrapped 15 mL centrifuge tube, add 6 mL of HBSS-HEPES assay buffer and supplement with 6 µL of 10% Pluronic F-127 and 6 µL of Fluo-4/AM solution. Briefly vortex the solution and allow to sit at room temperature, out of light, for 10 min.
      NOTE: To prevent photobleaching of Fluo-4, keep the dye out of light by wrapping aluminum foil around the centrifuge tube containing the dye solution and the lid of the assay plate.
  2. Prepare assay plates.
    1. Remove the assay plate from the incubator and confirm confluency using an inverted microscope. Manually remove the DMEM complete media by flicking it into a sink and blotting the top of the plate with a paper towel.
      NOTE: Medium is sufficiently removed by holding the plate horizontally and flicking, followed by rotating the plate 180°and repeating.
    2. Add HBSS-HEPES assay buffer plus probenecid solution to a 50 mL multi-channel pipette solvent reservoir.
      NOTE: This solution will be used for later washing steps and should be put aside and covered with aluminum foil until needed.
    3. Add 100 µL of the HBSS-HEPES assay buffer plus probenecid solution to each well with a multi-channel pipette and allow the cells to sit in the presence of probenecid for 5 min. Remove the HBSS-HEPES assay buffer by flicking into the sink in the same manner as in step 3.2.1.
    4. Add dye loading buffer to a separate 50 mL multi-channel pipette solvent reservoir. Add 50 µL of the dye loading buffer to each well of the assay plate using a multi-channel pipette. Incubate the plate for 45 min (37 °C, 5% CO2).
    5. While the plate is in the incubator, prepare the agonist/antagonist solutions.
      1. Antagonist solutions are stored as 31.6 mM solutions in DMSO. In a PCR tube, dilute 2 µL of the stock solution with 38 µL of 0.1% BSA/water to obtain a 1.58 mM solution. 2 µL of this solution will give a final concentration of 31.6 µM in the assay plate. Dilute 4 µL of the 1.58 mM antagonist solution with 36 µL of 5% DMSO/water (no BSA addition), and prepare the remaining concentrations through serial dilutions.
      2. Prepare a 108.3 µM (16.7x) solution of TFLLRN-NH2 in HBSS-HEPES assay buffer, which will give a final concentration of 6.5 µM when 6 µL is added to each well of the assay plate. For example, dissolve a 2 mg aliquot of TFLLRN-NH2 with 5.244 mL of HBSS-HEPES assay buffer to give a 0.5 mM stock solution. Mix 1.083 mL of this solution with 3.917 mL of HBSS-HEPES assay buffer to give a 108.3 µM solution.
        NOTE: TFLLRN-NH2 solutions should be stored in a -20 °C freezer when not in use.
    6. Remove the plate from the incubator and ensure dye uptake by using a fluorescence microscope at a suitable setting (typically for green fluorescent protein, see Figure 1 for an example of Fluo-4 successfully loaded into cells). Remove the dye loading buffer by flicking the plate in the same manner as step 3.2.1.
    7. Wash the cells 2x with 50 µL of the HBSS-HEPES assay buffer plus probenecid solution (from step 3.2.3) in each well (remove by flicking medium into the sink).
    8. Add 92 µL of HBSS-HEPES assay buffer to each well and again view the cells with a fluorescence microscope to ensure excess dye has been fully removed, and that cells remain adherent.
    9. Use a multi-channel pipette to add 2 µL of the antagonist solutions and vehicle to appropriate wells (see Table 1 for a representative plate map).

4. Performing the calcium mobilization assay

  1. Set the plate reader as follows: temperature: 37 °C; excitation wavelength: 485 nm; emission wavelength: 525 nm; measurement height: 7.8 mm (optimized); number of flashes: 100; number of repeats: 20.
  2. Incubate the plate for 15 min in the plate reader at 37 °C. Measure the background fluorescence in columns 1 and 2 (five scans per well).
  3. Eject the plate from the plate reader and quickly add 6 µL of agonist solution to each well in columns 1 and 2 from an 8-tube PCR strip using a multi-channel pipette.
    NOTE: For this step, we use an electronic 8-channel pipette with 0.1-10 µL pipette tips.
  4. Measure the change in fluorescence as calcium mobilization occurs in the cells. Perform 20 scans of each well according to the settings above.
    NOTE: Scanning each well in two columns 20x each takes approximately 5 min (i.e., ~15 s between scans of each well).
  5. Repeat steps 4.2-4.4 for columns 3-12 in two column increments.

5. Data analysis

  1. Export data from the assay as separate spreadsheets for each two-column group.
  2. Find the average of the basal fluorescence readings by using the function AVG(first scan: last scan). Do this for every well in both columns.
  3. Find the maximum fluorescence after the addition of agonist by using the function MAX(first scan: last scan).
  4. Calculate the change in fluorescence by subtracting the basal fluorescence from the maximum fluorescence.
  5. Subtract the change in fluorescence calculated for the negative control (addition of vehicle without agonist) from each well in the same column.
  6. Find the relative (normalized) change in fluorescence by dividing the change in fluorescence in each well by the change in fluorescence in the vehicle (0.1% DMSO/water) + 6.5 µM TFLLRN-NH2 well.
  7. Copy the non-control values, as percentages, into a statistics and graphing software program. If using the referenced software, choose the XY table option, with the Numbers chosen as the X-axis and replicate values in side-by-side subcolumns as the Y-axis.
  8. Use the program to plot concentration-response curves (CRCs) from the data. If using the referenced software, choose the log(inhibitor) vs. response - Variable slope (four parameters) option.

Results

The purpose of this assay is generally to produce concentration-response curves (CRCs) for three to four new parmodulins. On each assay plate, an additional CRC for a known compound, such as NRD-21, is often generated that acts as a quality check for the experiment due to its known IC50. To generate CRCs, a plate map such as the one depicted in Table 1 should be planned. If single-point concentration-responses are desired instead, compounds at 10 µM final concentrations (or other preferre...

Discussion

While the previously reported protocol72 was generally reliable and allowed us to identify a new lead parmodulin, NRD-21,62 a more efficient protocol was desired. The assay was further compromised during the supply shortage caused by the COVID-19 pandemic. Acquiring tips for the automated liquid handler became difficult, and attempting to wash, sterilize, and reuse the tips produced CRCs with significant variance. This facilitated an urgent series of experiments designed to...

Disclosures

C.D. is inventor on patents involving parmodulins and is the founder and a shareholder of Function Therapeutics, Inc., which is developing parmodulins for clinical use.

Acknowledgements

We thank Irene Hernandez, Trudy Holyst, Dr. Hartmut Weiler (Versiti Blood Research Institute), and Dr. Leggy Arnold (University of Wisconsin-Milwaukee) for providing space and indirect support of this project, and Dr. John McCorvy (Medical College of Wisconsin) for pertinent advice. We thank the National Heart, Lung, and Blood Institute (R15HL127636), the U.S. Dept. of Defense (W81XWH22101), and the National Science Foundation (2223225) for grant support.

Materials

NameCompanyCatalog NumberComments
Cell Culture Reagents
Adherent EA.hy926 cellsATCCCRL-2922
CellStripper cell dissociation reagentCorning25-056-CITrypsin can optionally be used, but should definitely be avoided with PAR2 assays.
Dulbecco's Modified Eagle Medium (DMEM) w/phenol redCorning10-013-CV
Fetal Bovine Serum (FBS)Avantor97068-091
Gelatin from porcine skinMilliporeSigmaG2500Use to make an aqueous 0.4% (w/v) solution with deionized water. Autoclave before use to sterilize.
Pen/Strep (100X)Corning30-002-CI
Phosphate-buffered saline (PBS)Corning21-040-CV
Trypan Blue (0.4% w/v)Corning25-900-CI
Calcium Mobilization Reagents
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)Thermo172571000
Bovine serum albumin (BSA)Avantor97061-420
Calcium chloride dihydrateThermo42352-0250
Dimethyl sulfoxideThermoJ66650-AD
Fluo-4/AMInvitrogenF14201
Hank's balanced salt solution (Ca/Mg/phenol-red free)Corning21-022-CV
Magnesium chloride hexahydrateMilliporeSigmaM2393
Pluronic F-127 (Poloxamer 407)Spectrum ChemicalP1166
ProbenecidTCI AmericaP1975
Sodium hydroxideVWR InternationalBDH9292
TFLLRN-NH2 (TFA salt)Prepared by Trudy Holyst at the Versiti Blood Research Institute
Materials
96-well culture-treated, black-walled, clear bottom assay plateCorning3603with transparent lids
Centrifuge tube, 15 mLAvantor89039-664
Centrifuge tube, 50 mLAvantor89039-656
Culture flask, T-75Corning353136tissue culture treated
Disposable reagent reservoir, 50 mLCorningRES-V-50-S
Enspire plate readerPerkin ElmerDiscontinued
Microcentrifuge tube, 1.5 mLAvantor20170-038
Pasteur pipette, 9"Fisher13-678-6Bmust be sterilized
PCR tube strip with separate flat cap stripsAvantor76318-802
Pipette tips, 20 µLBiotix63300042sterile, filtered tips
Pipette tips, 200 µLBiotix63300044sterile, filtered tips
Pipette tips, 1250 µLBiotix63300047sterile, filtered tips
PrismGraphPadvolume 6 used
Serological pipette, 5 mLTradewinds Direct 07-5005
Serological pipette, 10 mLTradewinds Direct 07-5010
Serological pipette, 25 mLTradewinds Direct 07-5025

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