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Screen Quest™ Fluo-8 No Wash Calcium Assay Kit

 Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 No Wash Calcium Assay Kit and Fluo-4 NW Calcium 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 cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Fluo-8 No Wash calcium assay kit or Fluo-4 NW kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (50µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations.
 Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 No Wash Calcium Assay Kit and Fluo-4 NW Calcium 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 cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Fluo-8 No Wash calcium assay kit or Fluo-4 NW kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (50µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations.
 Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 No Wash Calcium Assay Kit and Fluo-4 NW Calcium 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 cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Fluo-8 No Wash calcium assay kit or Fluo-4 NW kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (50µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations.
<strong>Generation of large cardiac tissue sheets (L-CTSs).</strong><br />(A) Optimal view of the L-CTS placed in a 10cm-sized UpCell dish. Scale bar = 1 mm. (B) (C) Calcium transient of L-CTSs. (B) Representative Fluo-8 image of L-CTS (see also S1 video) and region of interests (ROIs). Magnification &times;100. (C) Chronologic intensity change of Fluo-8. Note that the peak timings of 4 different ROIs are almost same chronologically. Source:&nbsp;<strong>Human iPS cell-derived cardiac tissue sheets for functional restoration of infarcted porcine hearts</strong> by Masanosuke Ishigami et al., <em>PLOS</em>, Aug 2018.
SLY-induced pore formation-dependent Ca<sup>2+</sup>&nbsp;influx triggers PNC formation. (A&ndash;C)&nbsp;rSLY induces Ca<sup>2+</sup>&nbsp;influx in human platelets. The purified platelets marked with Fluo-8 were resuspended in HBSS (with 2&thinsp;mM Ca<sup>2+</sup>) and rSLY/rSLY<sup>P353V</sup>&nbsp;(1&thinsp;&mu;g/mL) or rSLY that was pretreated by cholesterol (10&thinsp;&mu;g/mL). The Ca<sup>2+</sup>&nbsp;influx in platelets was observed using an FV1000 confocal laser scanning microscope. The Ca<sup>2+</sup>&nbsp;influx in platelets was observed using an FV1000 confocal laser scanning microscope. The following mean fluorescence intensity (MFI) of Ca<sup>2+</sup>&nbsp;mobilization was recorded by Series Analysis (XY-T) software in FV1000. C1-C10, cell 1-cell 10. (D) The EGTA effect on rSLY-induced CD62P release from platelets in human blood was assessed by flow cytometry. (E) The EGTA (3&thinsp;mM) effect on&nbsp;<em>S. suis</em>&nbsp;supernatant-induced PNC formation was detected by flow cytometry. THB and PBS are the negative controls for culture supernatant and proteins, respectively. EGTA was dissolved in H<sub>2</sub>O. Data in B and C are given as the mean&thinsp;&plusmn;&thinsp;SD of three independent experiments from three different blood donors. **<em>P</em>&thinsp;&lt;&thinsp;0.01; ns, no significance; Cho, cholesterol; rSLY, recombinant SLY; 05ZYH33, wild type strain; ∆sly, isogenic sly mutants; Sup, supernatant. Source: <strong>Suilysin-induced Platelet-Neutrophil Complexes Formation is Triggered by Pore Formation-dependent Calcium Influx </strong>by Zhang et al., <em>Scientific Reports</em>, Nov. 2016.
5-HT will attenuate Ca<sup>2+</sup> uptake under the normoxia condition. The fresh-made mitochondria were pre-incubated with Calcium indicator Quest Fluo-8<sup>TM</sup>, AM or membrane potential dye Rhodamine 123 for 15&thinsp;min and washed three times using potassium chloride (KCl) media containing 5&thinsp;mM succinate. After recording the baseline, the mitochondria were perfused with 10&thinsp;uM Ca<sup>2+</sup>&nbsp;or 5&thinsp;uM FCCP. (A) Serotonin hydrochloride (5-HT) decreases mitochondrial Ca<sup>2+</sup>&nbsp;uptake. (B) Serotonin creatinine sulfate (5-HT(H<sub>2</sub>SO<sub>4</sub>)) attenuate Ca<sup>2+</sup>&nbsp;uptake. (C) The percentage of Ca<sup>2+</sup>&nbsp;uptaken by mitochondria of 5-HT groups (n&thinsp;=&thinsp;6) and 5-HT (H<sub>2</sub>SO<sub>4</sub>) groups (n&thinsp;=&thinsp;6). (D) Ca<sup>2+</sup>&nbsp;induced mitochondrial Ca<sup>2+</sup>&nbsp;uptake can be inhibited by RUR (n&thinsp;=&thinsp;5). (E) Transmitochondrial potential (measured with 5&thinsp;nM Rh-123) in control and 100&thinsp;uM 5-HT pre-incubated mitochondria. 5&thinsp;uM FCCP (Protonophore trifluoromethoxy carbonyl cyanide phenylhydrazond) was added to depolarize the mitochondria (n&thinsp;=&thinsp;3). Source: <strong>5-HTR3 and 5-HTR4 located on the mitochondrial membrane and functionally regulated mitochondrial functions </strong>by Wang et al., <em>Scientific Reports</em>, Nov. 2016.
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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
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 AM 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 in growth medium with 1-5% FBS
  2. Add Fluo-8 NW dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
  3. Incubate at room temperature for 1 hour
  4. Monitor fluorescence intensity at Ex/Em = 490/525 nm

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. Fluo-8 NW stock solution:
Add 20 µL (for Cat. # 36314) or 200 µL (for Cat. # 36315 and # 36316) of DMSO into the vial of Fluo-8 NW (Component A), and mix them well. Note: 20 µL of Fluo-8 NW stock solution is enough for one plate. Un-used Fluo-8 NW stock solution can be aliquoted and 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. # 36314 (1 plate kit) and # 36315 (10 plates kit), make 1X assay buffer by adding 9 mL of HHBS (Component C) into 10X Pluronic® F127 Plus (1 mL, Component B), and mix them well.
b) For Cat. # 36316 (100 plates kit), make 1X assay buffer by adding the whole bottle of 10 X Pluronic® F127 Plus, (10 mL, Component B) 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. Aliquot and store un-used 1X assay buffer at < -20 oC. Protect from light and avoid repeated freeze-thaw cycles

PREPARATION OF WORKING SOLUTION

Fluo-8 NW dye-loading solution:
Add 20 µL of Fluo-8 NW stock solution into 10 mL of 1X assay buffer, and mix them well. Note: 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. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Fluo-8 NW dye-loading solution into the cell plate. [We offer 2 separate medium removal calcium assay kits (Cat.# 36308 and 36309) for those who prefer to keep the medium removal step].

  2. 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 oC, 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).

  3. Prepare the compound plate with HHBS or your desired buffer.

  4. 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 the experiment. Different instruments have their own intensity range. Adjust the signal test intensity to the level of 10% to 15% of the maximum instrument intensity counts. For example, the maximum fluorescence intensity count for FLIPR-384 is 65,000, so the instrument settings should be adjusted to have the 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 yield
Screen Quest™ Fluo-4 No Wash Calcium Assay Kit495528820000.161

Images


Citations


View all 228 citations: Citation Explorer
Cholecystokinin B receptor agonists alleviates anterograde amnesia in cholecystokinin-deficient and aged Alzheimer's disease mice
Authors: Zhang, Nan and Sui, Yixuan and Jendrichovsky, Peter and Feng, Hemin and Shi, Heng and Zhang, Xu and Xu, Shenghui and Sun, Wenjian and Zhang, Huatang and Chen, Xi and others,
Journal: Alzheimer's Research \& Therapy (2024): 1--16
Preclinical development of EXT608, an investigational parathyroid hormone derivative with extended half-life for the treatment of hypoparathyroidism
Authors: Hall, Daniel B and Kostyla, Caroline H and Hales, Laura M and Soliman, Tarik M
Journal: JBMR Plus (2024): ziae045
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
Arylsulfatases and neuraminidases modulate engagement of CCR5 by chemokines by removing key electrostatic interactions
Authors: Pinheiro, In{\^e}s and Calo, Nicolas and Paolini-Bertrand, Marianne and Hartley, Oliver
Journal: (2023)
Cholecystokinin B Receptor Agonists Alleviates Anterograde Amnesia in CCK-deficient and Aged Alzheimer's Disease Mice
Authors: Zhang, Nan and Sui, Yixuan and Jendrichovsky, Peter and Feng, Hemin and Shi, Heng and Zhang, Xu and Xu, Shenghui and Sun, Wenjian and Zhang, Huatang and Chen, Xi and others,
Journal: (2023)
Trans palmitoleic acid, a dairy fat biomarker, stimulates insulin secretion and activates G protein-coupled receptors with different a mechanism than cis isomer
Authors: Korkus, Eliza and Szustak, Marcin and Madaj, Rafal and Chworos, Arkadiusz and Drzazga, Anna and Kozio{\l}kiewicz, Maria and D{\k{a}}browski, Grzegorz and Czaplicki, Sylwester and Konopka, Iwona and Gendaszewska-Darmach, Edyta
Journal: Food \& Function (2023)
The insulinotropic activity of oleosomes prepared from various sea buckthorn cultivars in mouse and human pancreatic $\beta$ cell lines
Authors: Korkus, Eliza and Szustak, Marcin and D{\k{a}}browski, Grzegorz and Czaplicki, Sylwester and Kad{\l}ubowski, S{\l}awomir and Kozio{\l}kiewicz, Maria and Konopka, Iwona and Gendaszewska-Darmach, Edyta
Journal: NFS Journal (2023)
Optopharmacological tools for precise spatiotemporal control of oxytocin signaling in the central nervous system and periphery
Authors: Froemke, Robert and Ahmed, Ismail and Liu, Jingjing and Gieniec, Krystyna and Bair-Marshall, Chloe and Adewakun, Ayomiposi and Hetzler, Belinda and Arp, Christopher and Khatri, Latika and Vanwalleghem, Gilles and others,
Journal: (2023)
Protocol for high-throughput single-cell patterning using a reusable ultrathin metal microstencil
Authors: Tian, Qingqing and Xing, Kunming and Liu, Yongshu and Wang, Qian and Sun, Haonan and Sun, Ying-Nan and Zhang, Shusheng
Journal: STAR Protocols (2023): 102115
Progenitor-derived endothelin controls dermal sheath contraction for hair follicle regression
Authors: Martino, Pieter and Sunkara, Raghava and Heitman, Nicholas and Rangl, Martina and Brown, Alexia and Saxena, Nivedita and Grisanti, Laura and Kohan, Donald and Yanagisawa, Masashi and Rendl, Michael
Journal: Nature Cell Biology (2023): 1--13