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Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*

Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 NW Assay Kit and Fluo-4 NW Assay Kit. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar black 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™ Fluo 8-NW calcium assay kit or the Fluo-4 NW kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (25µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC50 of Carbachol using Fluo8 NW is about 1.2 µM.
Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 NW Assay Kit and Fluo-4 NW Assay Kit. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar black 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™ Fluo 8-NW calcium assay kit or the Fluo-4 NW kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (25µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC50 of Carbachol using Fluo8 NW is about 1.2 µM.
Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 NW Assay Kit and Fluo-4 NW Assay Kit. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar black 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™ Fluo 8-NW calcium assay kit or the Fluo-4 NW kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (25µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC50 of Carbachol using Fluo8 NW is about 1.2 µM.
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Fax1-800-609-2943
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Spectral properties
Correction Factor (260 nm)1.076
Correction Factor (280 nm)0.769
Extinction coefficient (cm -1 M -1)23430
Excitation (nm)495
Emission (nm)516
Quantum yield0.161
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Correction Factor (260 nm)
1.076
Correction Factor (280 nm)
0.769
Extinction coefficient (cm -1 M -1)
23430
Excitation (nm)
495
Emission (nm)
516
Quantum yield
0.161
Calcium flux assays are preferred methods in drug discovery for screening G protein coupled receptors (GPCR). Screen Quest™ Fluo-8 NW Calcium Assay Kit provides a homogeneous fluorescence-based assay for detecting the intracellular calcium mobilization. Cells expressing a GPCR of interest that signals through calcium are pre-loaded with our proprietary Fluo-8 NW which can cross cell membrane. Fluo-8 NW is the brightest calcium indicator available for HTS screening. Once inside the cell, the lipophilic blocking groups of Fluo-8 NW are cleaved by non-specific cell esterase, resulting in a negatively charged fluorescent dye that stays inside cells, and its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with screening compounds, the receptor signals release of intracellular calcium, which greatly increase the fluorescence of Fluo-8 NW. The characteristics of its long wavelength, high sensitivity, and 100-250 times fluorescence increases (when it forms complexes with calcium) make Fluo-8 NW an ideal indicator for measurement of cellular calcium. This Screen Quest Fluo-8 NW Calcium Assay 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.

Platform


Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff510 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode/Programmable liquid handling

Other instruments

ArrayScan, FDSS, FLIPR, FlexStation, IN Cell Analyzer, NOVOStar, ViewLux

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells
  2. Remove the growth medium
  3. Add Fluo-8 NW dye working solution
  4. Incubate at RT for 1 hour
  5. Monitor fluorescence intensity at Ex/Em = 490/525 nm

Important notes
Do not add additional probenecid. It is recommended to incubate the dye working solution no longer than 2 hours.
Thaw all components to room temperature before beginning protocol.

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. Fluo-8 NW stock solution:
For Cat No. 36307, add 10 µL of DMSO into Fluo-8 NW (Component A), and mix them well.
For Cat No. 36308 and 36309, add 100 µL of DMSO into Fluo-8 NW (Component A), and mix them well. Note: 10 µL of Fluo-8 NW stock solution is enough for 1 plate.

2. Assay Buffer stock solution (1X):
For Cat No. 36307 and 36308, add 9 mL of HHBS (Component C) into 10X Pluronic® F127 Plus (1 mL, Component B) and mix well.
For Cat No. 36309, add the whole bottle of 10X Pluronic® F127 Plus (10 mL, Component B) into 90 mL of HHBS buffer (not included in kit) and mix well. Note: 10 mL of 1X Assay Buffer is enough for one plate.

PREPARATION OF WORKING SOLUTION

Add 10 µL of Fluo-8 NW DMSO stock solution into 10 mL of 1X assay buffer and mix well. This working solution is stable for at least 2 hours at room temperature.

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 plate. Note: It is important to remove the growth medium in order to minimize background fluorescence and compound interference with serum or culture media. Note: Alternatively, grow the cells in growth medium with 0.5% - to 1% FBS to avoid medium removal step. In this case, 2X dye loading solution in HHBS buffer is needed. [We offer 2 separate no wash calcium assay kits (Cat No. 36315 and Cat No. 36316) for those who use 0.5% to 1% FBS in growth medium to avoid the medium removal step].

  2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Fluo-8 NW dye working solution into the cell plate.

  3. Incubate the dye-loading plate in a cell incubator for 30 minutes, and then incubate the plate at room temperature for another 30 minutes. Note: If the assay requires 37°C, perform the experiment immediately without further room temperature incubation. Note: If the cells can function well at room temperature for longer time, incubate the cell plate at room temperature for 1 - 2 hours (It is recommended that the incubation time be no longer than 2 hours.)

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

  5. Run the calcium flux assay by monitoring the fluorescence intensity at Ex/Em = 490/525 nm. Note: It is important to run the signal test before your experiment. Different instruments have their own intensity range. Adjust the signal test intensity to the level of 10% to 15% of the maximum intensity counts. For example, the maximum fluorescence intensity count for FLIPR-384 is 65,000, so the instrument setting should be adjusted to have its signal test intensity around 7,000 to 10,000.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)1.076
Correction Factor (280 nm)0.769
Extinction coefficient (cm -1 M -1)23430
Excitation (nm)495
Emission (nm)516
Quantum yield0.161

Images


Citations


View all 161 citations: Citation Explorer
Transient receptor potential vanilloid 1 interacts with TLR4/CD14 signaling pathway in lipopolysaccharide-mediated inflammation in macrophages
Authors: Hsu, Julia Chu-Ning and Tseng, Hsu-Wen and Chen, Chia-Hui and Lee, Tzong-Shyuan
Journal: Experimental Animals (2024): 23--0148
Structural basis of antibody inhibition and chemokine activation of the human CC chemokine receptor 8
Authors: Sun, Dawei and Sun, Yonglian and Janezic, Eric and Zhou, Tricia and Johnson, Matthew and Azumaya, Caleigh and Noreng, Sigrid and Chiu, Cecilia and Seki, Akiko and Arenzana, Teresita L and others,
Journal: Nature Communications (2023): 7940
The potent analgesia of intrathecal 2R, 6R-HNK via TRPA1 inhibition in LF-PENS-induced chronic primary pain model
Authors: Liu, An-Ran and Lin, Zhen-Jia and Wei, Ming and Tang, Yuan and Zhang, Hui and Peng, Xiang-Ge and Li, Ying and Zheng, Yu-Fan and Tan, Zhi and Zhou, Li-Jun and others,
Journal: The Journal of Headache and Pain (2023): 1--27
Renal tubular epithelial TRPA1 acts as an oxidative stress sensor to mediate ischemia-reperfusion-induced kidney injury through MAPKs/NF-$\kappa$B Signaling
Authors: Wu, Chung-Kuan and Wu, Chia-Lin and Lee, Tzong-Shyuan and Kou, Yu Ru and Tarng, Der-Cherng
Journal: International journal of molecular sciences (2021): 2309
Cellular repair mechanisms triggered by exposure to silver nanoparticles and ionic silver in embryonic zebrafish cells
Authors: Quevedo, Ana C and Lynch, Iseult and Valsami-Jones, Eugenia
Journal: Environmental Science: Nano (2021)
Lung epithelial TRPA1 mediates lipopolysaccharide-induced lung inflammation in bronchial epithelial cells and mice
Authors: Ko, Hsin-Kuo and Lin, An-Hsuan and Perng, Diahn-Warng and Lee, Tzong-Shyuan and Kou, Yu Ru
Journal: Frontiers in physiology (2020): 596314
MMP-12 activates protease-activated receptor-1, upregulates placenta growth factor, and leads to pulmonary emphysema
Authors: Hou, Hsin-Han and Wang, Hao-Chien and Cheng, Shih-Lung and Chen, Yen-Fu and Lu, Kai-Zen and Yu, Chong-Jen
Journal: American Journal of Physiology-Lung Cellular and Molecular Physiology (2018): L432--L442
UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells
Authors: Maschalidi, Sophia and Nunes-Hasler, Paula and Nascimento, Clarissa R and Sallent, Ignacio and Lannoy, Valérie and Garfa-Traore, Meriem and Cagnard, Nicolas and Sepulveda, Fern and o E , undefined and Vargas, Pablo and Lennon-Duménil, Ana-Maria and others, undefined
Journal: Nature Communications (2017): 1640
The establishment of appropriate methods for egg-activation by human PLCZ1 RNA injection into human oocyte
Authors: Yamaguchi, Takashi and Ito, Masahiko and Kuroda, Keiji and Takeda, Satoru and Tanaka, Atsushi
Journal: Cell Calcium (2017)
Screening for AMPA receptor auxiliary subunit specific modulators
Authors: Azumaya, Caleigh M and Days, Emily L and Vinson, Paige N and Stauffer, Shaun and Sulikowski, Gary and Weaver, C David and Nakagawa, Terunaga
Journal: PloS one (2017): e0174742

References


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Kinetic characterization of novel NR2B antagonists using fluorescence detection of calcium flux
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Amplitude distribution of calcium sparks in confocal images: theory and studies with an automatic detection method
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Nucleoplasmic and cytoplasmic differences in the fluorescence properties of the calcium indicator Fluo-3
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Detection of a trigger zone of bradykinin-induced fast calcium waves in PC12 neurites
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Improved four-color flow cytometry method using fluo-3 and triple immunofluorescence for analysis of intracellular calcium ion ([Ca2+]i) fluxes among mouse lymph node B- and T-lymphocyte subsets
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