Screen Quest™ Fura-2 No Wash Calcium Assay Kit
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Spectral properties
Excitation (nm) | 336 |
Emission (nm) | 505 |
Storage, safety and handling
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Related products
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See also: Calcium Indicators, Cell/Cytoplasmic Membrane Potential Activity & Analysis, Cell Signaling Pathways, Intracellular Ions, Ratiometric Calcium Indicators, Screen Quest™ Products
Excitation (nm) 336 | Emission (nm) 505 |
Calcium flux assays are preferred methods in drug discovery for screening G protein coupled receptors (GPCR). This ratiometric calcium assay kit allows homogeneous measurement of intracellular calcium changes caused by activation of G-protein-coupled receptors or calcium channels. Cells expressing a GPCR of interest that signals through calcium are pre-loaded with Fura-2 AM which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Fura-2 AM are cleaved by esterases, resulting in a negatively charged fluorescent dye that stays inside cells and its fluorescence wavelength is blue-shifted upon binding to calcium. When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which greatly increase the fluorescence intensity of Fura-2 at the short wavelength. The ratiometric characteristics of Fura-2 make this kit an ideal tool for more accurate measurement of cellular calcium concentration compared to Fluo-4 of the single wavelength. With a single addition, the assay is easy to perform and desirable in a high thoroughput environment. The assay can be used in a convenient 96-well or 384-well microtiter-plate format and readily adapted to automation.
Platform
Fluorescence microplate reader
Excitation | 340/380 nm |
Emission | 510 nm |
Cutoff | 470 nm |
Recommended plate | Black wall/Clear bottom |
Instrument specification(s) | Bottom read mode/Programmable liquid handling |
Other instruments
FDSS, FLIPR, FlexStationComponents
Example protocol
AT A GLANCE
Protocol Summary
- Prepare cells in growth medium
- Add Fura-2 AM dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
- Incubate at room temperature for 1-2 hour
- Monitor fluorescence intensity at Ex/Em = 340/510 nm and 380/510 nm
CELL PREPARATION
For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html
PREPARATION OF STOCK SOLUTIONS
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.
Note 20 µL of Fura-2 AM stock solution is enough for one plate. Unused Fura-2 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.
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.
1. Fura-2 AM stock solution
Add 200 µL of DMSO into the vial of Fura-2 AM (Component A), and mix them well.Note 20 µL of Fura-2 AM stock solution is enough for one plate. Unused Fura-2 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)
a) For Cat. # 36320 (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. # 36321 (100 plates kit), make 1X assay buffer by adding 90 mL of HHBS (Not included) into 10X Pluronic® F127 Plus (10 mL, Component B), 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
Fura-2 AM dye-loading solution
Add 20 µL of Fura-2 AM 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.
SAMPLE EXPERIMENTAL PROTOCOL
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Fura-2 AM dye-loading solution into the cell plate. Note: If your compounds interfere with the serum, then it is important to replace the growth medium with HHBS buffer.
- Incubate the dye-loading plate in a cell incubator for 1 hour, and then incubate the plate at room temperature for another 20 minutes. Note: If the assay requires 37 oC, perform the experiment immediately without further room temperature incubation.
- Prepare the compound plate with HHBS or your desired buffer.
- Run the calcium flux assay by monitoring the fluorescence increase at Ex/Em = 340/510 nm and 380/510 nm. Note: It is important to run the signal test before the experiment. Different instruments have their own intensity range. Note: For assays performed on FDSS, use the standard filters for Fura-2 calcium assays on the instrument.
Images

Figure 1. ATP dose response in CHO-K1 cells measured with Screen Quest™ Fura-2 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 Screen Quest™ Fura-2 No Wash Calcium Assay Kit for 1 hour at room temperature. ATP (50 µL/well) was added by a FlexStation (Molecular Devices) to achieve the final indicated concentrations.
Citations
View all 1 citations: Citation Explorer
Etablierung von zellbasierten Assays zur Identifizierung von Inhibitoren des Chemokins CXCL8
Authors: J{\"o}st, Marina
Journal: (2016)
Authors: J{\"o}st, Marina
Journal: (2016)
References
View all 119 references: Citation Explorer
Calcium imaging of cortical neurons using Fura-2 AM
Authors: Barreto-Chang OL, Dolmetsch RE.
Journal: J Vis Exp. (2009)
Authors: Barreto-Chang OL, Dolmetsch RE.
Journal: J Vis Exp. (2009)
Measurement of [Ca2+]i in whole cell suspensions using fura-2
Authors: Hirst RA, Harrison C, Hirota K, Lambert DG.
Journal: Methods Mol Biol (2006): 37
Authors: Hirst RA, Harrison C, Hirota K, Lambert DG.
Journal: Methods Mol Biol (2006): 37
Load of calcium probe Fura -2/AM in Escherichia coli cells
Authors: Shao M, Wang HM, Liu ZH, Shen P, Cai RX.
Journal: Wei Sheng Wu Xue Bao (2005): 805
Authors: Shao M, Wang HM, Liu ZH, Shen P, Cai RX.
Journal: Wei Sheng Wu Xue Bao (2005): 805
Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica
Authors: Froschauer EM, Kolisek M, Dieterich F, Schweigel M, Schweyen RJ.
Journal: FEMS Microbiol Lett (2004): 49
Authors: Froschauer EM, Kolisek M, Dieterich F, Schweigel M, Schweyen RJ.
Journal: FEMS Microbiol Lett (2004): 49
Photonic crystal fibre enables short-wavelength two-photon laser scanning fluorescence microscopy with fura-2
Authors: McConnell G, Riis E.
Journal: Phys Med Biol (2004): 4757
Authors: McConnell G, Riis E.
Journal: Phys Med Biol (2004): 4757
Abnormal spectra alteration observed in Triton calibration method for measuring [Ca2+]i with fluorescence indicator, fura-2
Authors: Xu T, Yang W, Huo XL, Song T.
Journal: J Biochem Biophys Methods (2004): 219
Authors: Xu T, Yang W, Huo XL, Song T.
Journal: J Biochem Biophys Methods (2004): 219
Cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (NSP4-EGFP) expressing cells loaded with Fura-2
Authors: Berkova Z, Morris AP, Estes MK.
Journal: Cell Calcium (2003): 55
Authors: Berkova Z, Morris AP, Estes MK.
Journal: Cell Calcium (2003): 55
Purinergic receptors have different effects in rat exocrine pancreas. Calcium signals monitored by fura-2 using confocal microscopy
Authors: Novak I, Nitschke R, Amstrup J.
Journal: Cell Physiol Biochem (2002): 83
Authors: Novak I, Nitschke R, Amstrup J.
Journal: Cell Physiol Biochem (2002): 83
AMPA-induced Ca(2+) influx in cultured rat cortical nonpyramidal neurones: pharmacological characterization using fura-2 microfluorimetry
Authors: Fischer W, Franke H, Scheibler P, Allgaier C, Illes P.
Journal: Eur J Pharmacol (2002): 53
Authors: Fischer W, Franke H, Scheibler P, Allgaier C, Illes P.
Journal: Eur J Pharmacol (2002): 53
Measurements of [Ca2+] using fura-2 in glioma C6 cells expressing calretinin with GFP as a marker of transfection: no Ca2+-buffering provided by calretinin
Authors: Billing-Marczak K, Przybyszewska M, Kuznicki J.
Journal: Biochim Biophys Acta (1999): 169
Authors: Billing-Marczak K, Przybyszewska M, Kuznicki J.
Journal: Biochim Biophys Acta (1999): 169
Application notes
A Comparison of Fluorescent Red Calcium Indicators for Detecting Intracellular Calcium Mobilization in CHO Cells
A Meta-Analysis of Common Calcium Indicators
A New Red Fluorescent & Robust Screen Quest™ Rhod-4™ Ca2+Indicator for Screening GPCR & Ca2+ Channel Targets
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
A Meta-Analysis of Common Calcium Indicators
A New Red Fluorescent & Robust Screen Quest™ Rhod-4™ Ca2+Indicator for Screening GPCR & Ca2+ Channel Targets
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
FAQ
Are there any calcium indicators that don't require probenecid (PBC)?
Are there upgraded trypan blue derivatives for cell viability testing?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
Do you offer any products for measuring intracellular calcium concentration or movement by flow cytometry?
Does EDTA inactivate proteinase K?
Are there upgraded trypan blue derivatives for cell viability testing?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
Do you offer any products for measuring intracellular calcium concentration or movement by flow cytometry?
Does EDTA inactivate proteinase K?