logo
AAT Bioquest

Screen Quest™ Fluo-4 No Wash Calcium Assay Kit

ATP Dose Response was measured in CHO-K1 cells with Screen Quest™ Fluo-4 No Wash Calcium Assay Kit. CHO-K1 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-4 No Wash calcium assay kit for 1 hour at room temperature. ATP (50µL/well) was added by Flexstation 3 (MDC) to achieve the final indicated concentrations.
ATP Dose Response was measured in CHO-K1 cells with Screen Quest™ Fluo-4 No Wash Calcium Assay Kit. CHO-K1 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-4 No Wash calcium assay kit for 1 hour at room temperature. ATP (50µL/well) was added by Flexstation 3 (MDC) to achieve the final indicated concentrations.
ATP Dose Response was measured in CHO-K1 cells with Screen Quest™ Fluo-4 No Wash Calcium Assay Kit. CHO-K1 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-4 No Wash calcium assay kit for 1 hour at room temperature. ATP (50µL/well) was added by Flexstation 3 (MDC) to achieve the final indicated concentrations.
Ordering information
Price
Catalog Number
Unit Size
Quantity
Add to cart
Additional ordering information
Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
ShippingStandard overnight for United States, inquire for international
Request quotation
Spectral properties
Extinction coefficient (cm -1 M -1)82000
Excitation (nm)495
Emission (nm)528
Quantum yield0.161
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
Related products
Screen Quest™ Membrane Potential Assay Kit *Orange Fluorescence*
Screen Quest™ Membrane Potential Assay Kit *Red Fluorescence*
Screen Quest™ 10X cell staining buffer with Phenol Red Plus™
Screen Quest™ 10X calcium assay buffer with Phenol Red Plus™
Screen Quest™ Luminometric Calcium Assay Kit
Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*
Screen Quest™ Fura-2 No Wash Calcium Assay Kit
Screen Quest™ Fluorimetric MDR Assay Kit
Screen Quest™ Colorimetric Chloride Channel Assay Kit
Screen Quest™ Colorimetric ELISA cAMP Assay Kit
Screen Quest™ Fluorimetric ELISA cAMP Assay Kit
Screen Quest™ TR-FRET No Wash cAMP Assay Kit
Screen Quest™ Live Cell cAMP Assay Service Pack
Screen Quest™ Fluorimetric Fatty Acid Uptake Assay Kit
Screen Quest™ Fluorimetric Glucose Uptake Assay Kit
Screen Quest™ Colorimetric Glucose Uptake Assay Kit
Screen Quest™ HEK-CNGC-Amylin 3 Receptor Cells
Screen Quest™ HEK-CNGC-Cannabinoid Receptor 1 Cells
Screen Quest™ CHO-Ga16- Chemokine (C-C) Receptor 2B Cells
Screen Quest™ HEK-CNGC-Chemokine (C-X-C motif) Receptor 4 Cells
Screen Quest™ HEK-CNGC-Dopamine Receptor 1 (DRD1) Cells
Screen Quest™ HEK-CNGC-Glucagon-like Receptor 1 (GLP1R) Cells
Screen Quest™ HEK-CNGC-Opiate Receptor-like 1 (ORL1) Cells
Screen Quest™ Human Nociceptin Receptor Ga16 coupled CHO Cells (NOP-Ga16)
Screen Quest™ CHO-Gqi Chimera Cell line
Screen Quest™ CHO-Gqo Chimera Cell line
Screen Quest™ CHO-Gqz Chimera Cell line
Screen Quest™ CHO-Gqs Chimera Cell line
Screen Quest™ CHO-Ga16 Chimera Cell line
Screen Quest™ No Wash Potassium Channel Assay Kit
Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit
Screen Quest™ Calbryte-590 Probenecid-Free and Wash-Free Calcium Assay Kit
Screen Quest™ Fluorimetric Neutrophil Elastase Inhibitor Screening Kit
Chemical Phosphorylation Reagent I (CPR I)
Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit *Red Fluorescence*
Cal Green™ 1, hexapotassium salt
Cal Green™ 1, AM [Equivalent to Calcium Green-1, AM]
Cal-590™-Dextran Conjugate *MW 3,000*
Cal-590™-Dextran Conjugate *MW 10,000*
Cal-590™ AM
Cal-590™, sodium salt
Cal-590™, potassium salt
Cal-630™ AM
Cal-630™, sodium salt
Cal-630™, potassium salt
Cal-630™-Dextran Conjugate *MW 3,000*
Cal-630™-Dextran Conjugate *MW 10,000*
Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*
Fluo-4, Pentapotassium Salt
Cal Red™ R525/650 potassium salt
Cal Red™ R525/650 AM
Cal-520®-Dextran Conjugate *MW 3,000*
Cal-520®-Dextran Conjugate *MW 10,000*
Cal-520®-Biotin Conjugate
Cal-520®-Biocytin Conjugate
Cal-520® NHS Ester
Cal-520® maleimide
Fluo-3, AM *CAS 121714-22-5*
Fluo-3, AM *UltraPure grade* *CAS 121714-22-5*
Fluo-3, AM *Bulk package* *CAS 121714-22-5*
Fluo-3FF, AM *UltraPure grade* *Cell permeant*
Fluo-3, pentasodium salt
Fluo-3, pentapotassium salt
Fluo-3, pentaammonium salt
Fluo-3FF, pentapotassium salt
Fluo-8®, AM
Fluo-8®, sodium salt
Fluo-8®, potassium salt
Fluo-8H™, AM
Fluo-8H™, sodium salt
Fluo-8L™, AM
Fluo-8L™, sodium salt
Fluo-8L™, potassium salt
Fluo-8FF™, potassium salt
Fluo-8FF™, AM
Cal-520®, AM
Cal-520®, sodium salt
Cal-520®, potassium salt
Cal-520FF™, AM
Cal-520FF™, potassium salt
Mag-Fluo-4 potassium salt
Mag-Fluo-4 AM
Fluo-2, potassium salt
Fluo-2, AM
Fluo-5F, AM *Cell permeant*
Fluo-5F, pentapotassium Salt *Cell impermeant*
Fluo-5N, AM *Cell permeant*
Fluo-5N, pentapotassium Salt *Cell impermeant*
Cal-520N™, AM
Cal-520N™, potassium salt
Calbryte™ 520 AM
Calbryte™ 520, potassium salt
Calbryte™ 590 AM
Calbryte™ 590, potassium salt
Calbryte™ 630 AM
Calbryte™ 630, potassium salt
Calbryte™-520L AM
Calbryte™-520L, potassium salt
Cal-500™, potassium salt
Cal-500™ AM
Cal-670™, potassium salt
Cal-670™-Dextran Conjugate *MW 3,000*
Cal-670™-Dextran Conjugate *MW 10,000*
Cal-770™, potassium salt
Cal-770™-Dextran Conjugate *MW 3,000*
Cal-770™-Dextran Conjugate *MW 10,000*
Calbryte™-520XL azide
Calbryte™-520XL, potassium salt
Calbryte™-520XL AM
Calbryte™-520XL-Dextran
Cal-520L®-Dextran Conjugate *MW 10,000*
RatioWorks™ Cal-520L®/Cy5-Dextran Conjugate *MW 10,000*
Cal-520L™ maleimide
RatioWorks™ Cal-520®/zFluor 647™ -Dextran Conjugate *MW 10,000*
Cal-590L® Dextran Conjugate *MW 10,000*
RatioWorks™ Cal-590L®/Cy5-Dextran Conjugate *MW 10,000*
Cal-520® amine
Cal-520® azide
Cal-520® alkyne
Cal-520ER™ potassium salt
Cal-520ER™ AM
Show More (111)

OverviewpdfSDSpdfProtocol


Extinction coefficient (cm -1 M -1)
82000
Excitation (nm)
495
Emission (nm)
528
Quantum yield
0.161
Calcium flux assays are preferred methods in drug discovery for screening G protein coupled receptors (GPCR). Screen Quest™ Fluo-4 No Wash 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 Fluo-4 AM which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Fluo-4 AM are cleaved by non-specific cell esterase, resulting in a negatively charged Fluo-4 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-4. This Screen Quest Fluo-4 No Wash 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
Emission525nm
Cutoff515 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
  2. Add Fluo-4 AM dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
  3. Incubate at 37°C for 1 hour
  4. Monitor fluorescence intensity at Ex/Em = 490/525 nm

Important notes
Do not add additional probenecid. Thaw all the kit components at room temperature before use.

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-4 AM stock solution:
Add 200 µL of DMSO into the vial of Fluo-4 AM (Component A), and mix them well. Note: 20 µL of Fluo-4 NW stock solution is enough for one plate. Unused Fluo-4 AM 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):
Make 1X Assay Buffer by adding 1 mL of 10X Pluronic® F127 Plus (10 mL, Component B) into 9 mL of HHBS buffer (Component C) and mix well. Note: 10 mL of 1X assay buffer is enough for one plate. Aliquot and store un-used 10X Pluronic® F127 Plus (Component B) at < -20 oC. Protect from light and avoid repeated freeze-thaw cycles.

PREPARATION OF WORKING SOLUTION

Fluo-4 AM dye-loading solution:
Add 20 µL of Fluo-4 NW 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. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Fluo-4 AM dye-loading solution into the cell plate.

  2. Incubate the dye-loading plate in a cell incubator for 1 hour, and then incubate the plate at room temperature for another 15 to 30 minutes. Note: If the assay requires 37°C, perform the experiment immediately without further room temperature incubation.

  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.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)82000
Excitation (nm)495
Emission (nm)528
Quantum yield0.161

Product Family


Images


Citations


View all 1 citations: Citation Explorer
Lysophosphatidylcholine and its phosphorothioate analogues potentiate insulin secretion via GPR40 (FFAR1), GPR55 and GPR119 receptors in a different manner
Authors: Drzazga, Anna and Kristinsson, Hjalti and Salaga, Maciej and Zatorski, Hubert and Koziolkiewicz, Maria and Gendaszewska-Darmach, Edyta and Bergsten, Peter
Journal: Molecular and Cellular Endocrinology (2017)