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

Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit

Optimized for Difficult Cell Lines

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.

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.

Protocol

COO

COA

Catalog	Size	Price
36307	1 Plate	Price
36308	10 Plates	Price
36309	100 Plates	Price

Components

Catalog 36307 (1 Plate)

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

Catalog 36308 (10 Plates)

Component A: Fluo-8 NW	1 vial, lyophilized
Component B: 10X Pluronic® F127 Plus	10 bottles (1 mL/bottle)
Component C: HHBS (Hanks' buffer with 20 mM Hepes)	1 bottle (100 mL)

Catalog 36309 (100 Plates)

Component A: Fluo-8 NW	10 vials, lyophilized
Component B: 10X Pluronic® F127 Plus	10 bottles (10 mL/bottle)
Component C: HHBS (Hanks' buffer with 20 mM Hepes)	Not included

Citations

View all 163 citations: Citation Explorer

COVID-19-associated neuroinflammation and astrocyte death in the brain linked to ORF3a-induced activation of Sur1-mediated ion channels

Authors:

Gerzanich, Volodymyr and Zhang, Chenyu and Zhang, Jiantao and Sallapalli, Bhargava Teja and Pei, Shaokai and Nasr, Mohamed and Tosun, Cigdem and Zhang, Yanjin and Tang, Qiyi and Simard, J Marc and others,

Journal:

mBio (2025): e02012--25

Piezo1 promotes the progression of necrotizing enterocolitis by activating the Ca2 (+)/CaMKII-dependent pathway

Authors:

Li, Zhenyu and Jiang, Qinlei and Wei, Jiaqi and Dang, Dan and Meng, Zhaoli and Wu, Hui

Journal:

Communications Biology (2025): 417

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

References

View all 26 references: Citation Explorer

Novel fluo-4 analogs for fluorescent calcium measurements

Authors:

Martin VV, Beierlein M, Morgan JL, Rothe A, Gee KR.

Journal:

Cell Calcium (2004): 509

Kinetic characterization of novel NR2B antagonists using fluorescence detection of calcium flux

Authors:

Bednar B, Cunningham ME, Kiss L, Cheng G, McCauley JA, Liverton NJ, Koblan KS.

Journal:

J Neurosci Methods (2004): 247

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41

Authors:

do Ceu Monteiro M, Sansonetty F, Goncalves MJ, O'Connor JE.

Journal:

Cytometry (1999): 302

Amplitude distribution of calcium sparks in confocal images: theory and studies with an automatic detection method

Authors:

Cheng H, Song LS, Shirokova N, Gonzalez A, Lakatta EG, Rios E, Stern MD.

Journal:

Biophys J (1999): 606

A simple numerical model of calcium spark formation and detection in cardiac myocytes

Authors:

Smith GD, Keizer JE, Stern MD, Lederer WJ, Cheng H.

Journal:

Biophys J (1998): 15

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 yield	0.16 ¹

Storage, safety and handling

Certificate of Origin	Download PDF
H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Instrument settings

Fluorescence microplate reader
Excitation	490 nm
Emission	525 nm
Cutoff	510 nm
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Bottom read mode/Programmable liquid handling

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

Telephone
Fax
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Technical Support	Contact us
Request quotation	Request
Purchase order	Send to sales@aatbio.com
Shipping	Standard overnight for United States, inquire for international

Page updated on October 13, 2025