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Screen Quest™ Luminometric Calcium Assay Kit

ATP Dose Response on CHO-aeq cells. CHO cells stably transfected with apoaequrin were seeded overnight at 50,000 cells/100 µL/well in a Costar white wall/clear bottom 96-well plate. The growth medium was removed and the cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Coelenterazine Calcium Assay Kit for 3 hours at room temperature and protected from light. ATP (25 µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC50 of ATP is about 0.8 µM.
ATP Dose Response on CHO-aeq cells. CHO cells stably transfected with apoaequrin were seeded overnight at 50,000 cells/100 µL/well in a Costar white wall/clear bottom 96-well plate. The growth medium was removed and the cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Coelenterazine Calcium Assay Kit for 3 hours at room temperature and protected from light. ATP (25 µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC50 of ATP is about 0.8 µM.
ATP Dose Response on CHO-aeq cells. CHO cells stably transfected with apoaequrin were seeded overnight at 50,000 cells/100 µL/well in a Costar white wall/clear bottom 96-well plate. The growth medium was removed and the cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Coelenterazine Calcium Assay Kit for 3 hours at room temperature and protected from light. ATP (25 µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC50 of ATP is about 0.8 µM.
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OverviewpdfSDSpdfProtocol


Calcium flux assays are preferred methods in drug discovery for screening G protein coupled receptors (GPCR). This kit uses a highly calcium-sensitive and membrane-permeable coelenterazine analog as a calcium indicator for the cells that are transfected with apoaequorin gene. Aequorin is a calcium-sensitive bioluminescent protein from the jellyfish Aequorea victoria that has been used extensively as a calcium indicator in cells. The aequorin complex emits blue light when bound to calcium ions. The luminescence intensity is directly proportional to the Ca2+ concentration. Our coelenterazine-based kit is much more sensitive than the fluorescence-based calcium assay kits (such as Fluo-4, Fluo-3, Calcium-3 and Calcium-4). This kit provides an optimized assay method for monitoring G-protein-coupled receptors (GPCRs) and calcium channels. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation. It might be useful for monitoring of intracellular calcium mobilization in a specified compartment given that recombinant apoaequorin proteins can now be targeted to specific organelles, cells and tissues.

Platform


Luminescence microplate reader

Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode/Programmable liquid handling

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells by removing growth medium
  2. Add Coelenterazine-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
  3. Incubate at room temperature for 3-4 hours
  4. Monitor aequorin luminescence intensity

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Coelenterazine analog:
Add 250 µL of 100% ETOH (Component B) into the vial of Coelenterazine analog (Component A), and mix them well. Note: 25 µL of reconstituted coelenterazine analog is enough for one plate. Unused coelenterazine analog stock solution can be stored at < -20 oC for more than one month if the tubes are sealed tightly. Protect from light and avoid repeated freeze-thaw cycles.

2. Assay Buffer (1X):
a) For Cat. # 36305 (10 plates kit), ready to use 1X Assay Buffer (Component C).
b)For Cat. # 36306 (100 plates kit), make 1X assay buffer by diluting 10 mL of 10X Assay Buffer (Component C) into 90 mL of HHBS buffer (not included in the kit), and mix them well. Note: 10 mL of 1X assay buffer is enough for one plate. Store unused 1X assay buffer at 4 oC.

PREPARATION OF WORKING SOLUTION

Coelenterazine-loading solution:
Add 25 µL of ETOH reconstituted coelenterazine analog (Prepartion Of Stock Solutions) into 10 mL of 1X assay buffer (Preparation Of Stock Solutions), and mix them well. Note: This working solution is stable for at least 2 hours at room temperature, protected from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

  1. Remove the growth medium from the cell plates. Note: It is important to remove the growth medium in order to minimize compound interference with serum or culture media. Note: Alternatively, grow the cells in growth medium with 0.5-1% FBS to avoid medium removal step. In this case, 2X Coelenterazine-loading solution in 1X buffer is needed.

  2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) Coelenterazine-loading solution into the cell plates.

  3. Incubate the Coelenterazine-loading plates at room temperature for 3-4 hours, protected from light.

  4. Prepare the compound plates with HHBS or the desired buffer.

  5. Monitor the aequorin luminescence intensity by using the photon detection system that has an enclosed chamber containing a photomultiplier. The instrument must completely exclude outside light.

Images


Citations


View all 3 citations: Citation Explorer
Intracellular Ca2+ is important for flagellin-triggered defense in Arabidopsis and involves inositol polyphosphate signaling
Authors: Ma, Yi and Zhao, Yichen and Berkowitz, Gerald A
Journal: Journal of Experimental Botany (2017)
Bioluminescence Resonance Energy Transfer (BRET) Assay for Determination of Molecular Interactions in Living Cells
Authors: Harikumar, Kaleeckal G and Yan, Yan and Xu, Ting-Hai and Melcher, Karsten and Xu, H Eric and Miller, Laurence J
Journal: (2017)
Orchestration of salivary secretion mediated by two different dopamine receptors in the blacklegged tick Ixodes scapularis
Authors: Kim, Donghun and Simo, Ladislav and Park, Yoonseong
Journal: Journal of Experimental Biology (2014): 3656--3663

References


View all 150 references: Citation Explorer
Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development
Authors: Cheung CY, Webb SE, Meng A, Miller AL.
Journal: Int J Dev Biol (2006): 561
Mitochondrial adaptations within chronically ischemic swine myocardium
Authors: McFalls EO, Sluiter W, Schoonderwoerd K, Manintveld OC, Lamers JM, Bezstarosti K, van Beusekom HM, Sikora J, Ward HB, Merkus D, Duncker DJ.
Journal: J Mol Cell Cardiol (2006): 980
Blue fluorescent protein from the calcium-sensitive photoprotein aequorin: catalytic properties for the oxidation of coelenterazine as an oxygenase
Authors: Inouye S, Sasaki S.
Journal: FEBS Lett (2006): 1977
Self-illuminating quantum dots light the way
Authors: Frangioni JV., undefined
Journal: Nat Biotechnol (2006): 326
Expression, purification and characterization of calcium-triggered luciferin-binding protein of Renilla reniformis
Authors: Inouye S., undefined
Journal: Protein Expr Purif. (2006)
In vivo testing of Renilla luciferase substrate analogs in an orthotopic murine model of human glioblastoma
Authors: Otto-Duessel M, Khankaldyyan V, Gonzalez-Gomez I, Jensen MC, Laug WE, Rosol M.
Journal: Mol Imaging (2006): 57
Crystal structure of obelin after Ca2+-triggered bioluminescence suggests neutral coelenteramide as the primary excited state
Authors: Liu ZJ, Stepanyuk GA, Vysotski ES, Lee J, Markova SV, Malikova NP, Wang BC.
Journal: Proc Natl Acad Sci U S A (2006): 2570
Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells
Authors: Pfleger KD, Dromey JR, Dalrymple MB, Lim EM, Thomas WG, Eidne KA.
Journal: Cell Signal (2006): 1664
Luminescence of imidazo[1,2-a]pyrazin-3(7H)-one compounds
Authors: Teranishi K., undefined
Journal: Bioorg Chem. (2006)
Role of molecular oxygen in the bioluminescence of the firefly squid, Watasenia scintillans
Authors: Tsuji FI., undefined
Journal: Biochem Biophys Res Commun (2005): 250