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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.
Ordering information
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Catalog Number36307
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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ShippingStandard overnight for United States, inquire for international
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

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


Component A: Fluo-8 NW1 vial, lyophilized
Component B: 10X Pluronic® F127 Plus1 bottle (1 mL)
Component C: HHBS (Hanks' buffer with 20 mM Hepes)1 bottle (9 mL)

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

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
Screen Quest™ Fluo-8 No Wash Calcium Assay Kit495516234300.1611.0760.769

Citations


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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
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Authors: SHIOMI, YOSHIHIRO and YOSHIMURA, MAKOTO and KUKI, KAZUMASA and HORI, YUKO and TANAKA, TAKAO
Journal: Anticancer Research (2017): 4127--4137
Laminarin counteracts diet-induced obesity associated with glucagon-like peptide-1 secretion
Authors: Yang, Liusong and Wang, Lina and Zhu, Canjun and Wu, Junguo and Yuan, Yexian and Yu, Lulu and Xu, Yaqiong and Xu, Jingren and Wang, Tao and Liao, Zhengrui and others, undefined
Journal: Oncotarget (2017): 99470
2-OMe-lysophosphatidylcholine analogues are GPR119 ligands and activate insulin secretion from βTC-3 pancreatic cells: Evaluation of structure-dependent biological activity
Authors: Drzazga, Anna and Sowińska, Agata and Krzemińska, Agnieszka and Okruszek, Andrzej and Paneth, Piotr and Koziolkiewicz, Maria and Gendaszewska-Darmach, Edyta
Journal: Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids (2017)
L-Type Calcium Channel Inhibition Contributes to the Proarrhythmic Effects of Aconitine in Human Cardiomyocytes
Authors: Wu, Jianjun and Wang, Xiangchong and Chung, Ying Ying and Koh, Cai Hong and Liu, Zhenfeng and Guo, Huicai and Yuan, Qiang and Wang, Chuan and Su, Suwen and Wei, Heming
Journal: PloS one (2017): e0168435

References


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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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